WO2000026401A1 - METHYLATED CpG ISLAND AMPLIFICATION (MCA) - Google Patents

METHYLATED CpG ISLAND AMPLIFICATION (MCA) Download PDF

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Publication number
WO2000026401A1
WO2000026401A1 PCT/US1999/025251 US9925251W WO0026401A1 WO 2000026401 A1 WO2000026401 A1 WO 2000026401A1 US 9925251 W US9925251 W US 9925251W WO 0026401 A1 WO0026401 A1 WO 0026401A1
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nucleic acid
seq
cell
methylation
mintl
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PCT/US1999/025251
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French (fr)
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Jean-Pierre Issa
Stephen Baylin
Minoru Toyota
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The Johns Hopkins University School Of Medicine
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Priority to CA002350085A priority Critical patent/CA2350085A1/en
Priority to EP99971462A priority patent/EP1127157A4/en
Priority to JP2000579773A priority patent/JP2002533061A/en
Priority to AU14537/00A priority patent/AU774774B2/en
Publication of WO2000026401A1 publication Critical patent/WO2000026401A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • the present invention relates generally to regulation of gene expression and more specifically to a method of determining the DNA methylation status of CpG sites in a given locus.
  • DNA methylases transfer methyl groups from the universal methyl donor S-adenosyl methionine to specific sites on the DNA.
  • Several biological functions have been attributed to the methylated bases in DNA.
  • the most established biological function for methylated DNA is the protection of DNA from digestion by cognate restriction enzymes.
  • the restriction modification phenomenon has, so far, been observed only in bacteria.
  • Mammalian cells possess a different methylase that exclusively methylates cytosine residues that are 5' neighbors of guanine (CpG). This modification of cytosine residues has important regulatory effects on gene expression, especially when involving CpG rich areas, known as CpG islands, located in the promoter regions of many genes.
  • Methylation has been shown by several lines of evidence to play a role in gene activity, cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes (Razin, A., H., and Riggs, R.D. eds. in DNA Methylation Biochemistry and Biological Significance. Springer- Verlag, New York, 1984). Methylation has been shown by several lines of evidence to play a role in gene activity, cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes (Razin, A. H. and Riggs, R.D. eds. in DNA Methylation Biochemistry and Biological Significance. Springer-Verlag. New York.
  • VHL gene showed aberrant methylation in a subset of sporadic renal cell carcinomas (Herman, et al, Proc. Natl. Acad. Sci., U.S.A., 91 :9700. 1994).
  • a tumor suppressor gene can also be abolished by de novo DNA methylation of a normally unmethylated CpG island (Issa, et al, Nature Genet., 2:536, 1994; Herman, et al, supra; Merlo, et al, Nature Med, 1:686, 1995; Herman, et al, Cancer Res., 56:722, 1996; Graff, et al, Cancer Res., 5 5:5195, 1995; Herman, et al, Cancer Res., 5_£:4525. 1995).
  • Human cancer cells typically contain somatically altered nucleic acid, characterized by mutation, amplification, or deletion of critical genes.
  • nucleic acid from human cancer cells often displays somatic changes in DNA methylation (E.R. Fearon, et al, Cell, 61:759, 1990;
  • CGI CpG island
  • CGIs are short sequences rich in the CpG dinucleotide and can be found in the 5' region of about half of all human genes (Bird, 1986). Methylation of cytosine within 5' CGIs is associated with loss of gene expression and has been seen in physiological conditions such as X chromosome inactivation and genomic imprinting (reviewed in Latham, 1996). Aberrant methylation of CGIs has been detected in genetic diseases such as the fragile-X syndrome (Hansen et al, 1992), in aging cells (Issa et al, 1994) and in neoplasia.
  • TSG methylation in cancer is usually associated with (1) lack of gene transcription and (2) absence of coding region mutation.
  • CGI methylation serves as an alternative mechanism of gene inactivation in cancer.
  • CGI methylation in human cancers remain poorly defined. Aging could play a factor in this process because methylation of several CGIs could be detected in an age-related manner in normal colon mucosa as well as in CRC (Issa et al, 1994).
  • aberrant methylation of CGIs has been associated with the MI phenotype in CRC (Ahuja et al, 1997) as well as specific carcinogen exposures (Issa et al, 1996).
  • an understanding of aberrant methylation in CRC has been somewhat limited by the small number of CGIs analyzed to date.
  • Mapping of methylated regions in DNA has relied primarily on Southern hybridization approaches, based on the inability of methylation- sensitive restriction enzymes to cleave sequences which contain one or more methylated CpG sites. This method provides an assessment of the overall methylation status of CpG islands, including some quantitative analysis, but is relatively insensitive and requires large amounts of high molecular weight DNA.
  • Another method utilizes bisulfite treatment of DNA to convert all unmethylated cytosines to uracil.
  • the altered DNA is amplified and sequenced to show the methylation status of all CpG sites.
  • this method is technically difficult, labor intensive and without cloning amplified products, it is less sensitive than Southern analysis, requiring approximately 10% of the alleles to be methylated for detection.
  • the invention provides a method for detecting a methylated CpG- containing nucleic acid. This method can be used to identify sequences which are differentially methylated during a disease process such as a cell proliferative disorder.
  • a method for identifying a methylated CpG-containing nucleic acid.
  • the method includes contacting a nucleic acid sample suspected of containing a CpG-containing nucleic acid, with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites, under conditions and for a time to allow cleavage of unmethylated nucleic acid; and contacting the sample with an isoschizomer of the methylation sensitive restriction endonuclease, wherein the isoschizomer of the methylation sensitive restriction endonuclease cleaves both methylated and unmethylated CpG sites.
  • Oligonucleotides are added to the nucleic acid sample under conditions and for a time to allow ligation of the oligonucleotides to nucleic acid cleaved by the restriction endonuclease and the digested nucleic acid is amplified for further analysis.
  • a method for detecting an age- associated disorder associated with methylation of CpG islands in a nucleic acid sequence of interest in a subject having or at risk of having said disorder.
  • the method includes contacting a nucleic acid sample suspected of comprising a CpG-containing nucleic acid with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites under conditions and for a time to allow cleavage of unmethylated nucleic acid, and contacting the sample with an isoschizomer of the methylation sensitive restriction endonuclease, wherein the isoschizomer of the methylation sensitive restriction endonuclease cleaves both methylated and unmethylated CpG sites.
  • Oligonucleotides are added to the nucleic acid sample under conditions and for a time to allow ligation of the oligonucleotides to nucleic acid cleaved by the restriction endonuclease, and the digested nucleic acid is amplified.
  • the amplified, digested nucleic acid is contacted with a membrane and the membrane is hybridized with a probe of interest.
  • a method for evaluating the response of a cell to an agent. The method includes contacting a nucleic acid sample suspected of containing a CpG-containing nucleic acid with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites, under conditions and for a time to allow cleavage of unmethylated nucleic acid, and contacting the sample with an isoschizomer of the methylation sensitive restriction endonuclease, wherein the isoschizomer of the methylation sensitive restriction endonuclease cleaves both methylated and unmethylated CpG sites.
  • Oligonucleotides are added to the nucleic acid sample under conditions and for a time to allow ligation of the oligonucleotides to nucleic acid cleaved by the restriction endonuclease, and the digested nucleic acid is amplified.
  • the amplified, digested nucleic acid is adhered to a membrane and the membrane is hybridized with a probe of interest.
  • kits for the detection of a methylated CpG- containing nucleic acid includes a carrier means containing one or more containers including a container containing an oligonucleotide for ligation of the oligonucleotides to nucleic acid, a second container containing a methylation sensitive restriction endonuclease and a third container containing an isoschizomer of the methylation sensitive endonuclease.
  • the kit includes a carrier means containing one or more containers containing a membrane, wherein the membrane has a member of the group consisting SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 (MINT1, MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINT14, MINT15, MINT17, MINT19, MINT20, MINT22, MINT23,
  • an isolated nucleic acid including a member selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 (MINT1, MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINT14, MINT15, MINT17, MINT19, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32, and MINT33) is provided.
  • An isolated methylated nucleic acid sequence having a sequence as set forth in a member of the group consisting of SEQ ID NO
  • FIG. 1 is a schematic diagram of MCA.
  • a hypothetical fragment of genomic DNA is represented by a solid line, with 7 Smal sites depicted by tick marks. Methylated Smal sites are indicated by an m. Fragments B and D are CpG islands. B is methylated in both normal (right) and cancer (left), while D is differentially methylated in cancer.
  • unmethylated Smal sites are eliminated by digestion with Smal (which is methylation-sensitive and does not cleave when its recognition sequence CCCGGG contains a methylated CpG), which leaves the fragment blunt ended.
  • Methylated Smal sites are then digested with the non-methylation sensitive Smal isoschizomer Xmal, which digests methylated CCCGGG sites, leaving a CCGG overhang (sticky ends). Adaptors are ligated to these sticky ends, and PCR is performed to amplify the methylated sequences.
  • the MCA amplicons can be used directly in a dot blot analysis to study the methylation status of any gene for which a probe is available (left). Alternatively, MCA products can be used to clone differentially methylated sequence by RDA (right).
  • FIG. 2 shows an the nucleotide sequence of a differentially Methylated Clone, MINT2 obtained by MCA Followed by RDA.
  • the restriction endonuclease sites for Smal are underlined.
  • Primer sequences used for bisulfite-PCR are also underlined.
  • the restriction endonuclease site for BstUI used to detect methylation after bisulfite PCR is shown by a gray box.
  • FIG. 3 show a map of the versican gene first exon (filled box) and flanking regions.
  • the position of MINTl 1 is shown by a solid line (on top). CpG sites are indicated below. Location of the primers used for bisulfite-PCR are shown by arrows.
  • FIG. 4 is a pictorial representation of global hypermethylation in CRC. Each column represents a separate gene locus. Each row is a primary colorectal cancer (samples above the bold solid line) or polyp ( below the bold solid line). Black squares: methylation > 10%. Gray squares: 1-10% methylation. White squares: ⁇ 1% methylation.
  • A-D are cancers. E and F are adenomas. MI denotes the presence of microsatellite instability. ND, not done.
  • FIG. 5 shows a model integrating CGI methylation in colorectal carcinogenesis.
  • FIGS. 6A-H are the nucleic acid sequences of MINT1-33 (SEQ ID NO: 1-33).
  • the present invention provides a method for identifying a methylated CpG-containing nucleic acid called methylated CpG island amplification (MCA).
  • MCA can be used to study methylation in normal and neoplastic cells, and allows rapid screening of nucleic acid samples for the presence of hypermethylation of specific genes.
  • MCA can also be used to clone genes and nucleic acid sequences differentially methylated in normal and abnormal tissues and cells.
  • nucleic acid sample in purified or nonpurified form, can be utilized as the starting nucleic acid or acids, provided it contains, or is suspected of containing, a nucleic acid sequence containing the target locus (e.g., CpG-containing nucleic acid).
  • the CpG-containing nucleic acid will be DNA.
  • the process may employ, for example, samples that contain DNA, or DNA and RNA, including messenger RNA, wherein DNA or RNA may be single stranded or double stranded, or a DNA-RNA hybrid may be included in the sample.
  • a mixture of nucleic acids may also be employed.
  • the specific nucleic acid sequence to be detected may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be studied be present initially in a pure form; the nucleic acid may be a minor fraction of a complex mixture, such as contained in whole human DNA.
  • the nucleic acid may be contained in a biological sample. Such samples include but are not limited to a serum, urine, saliva, cerebrospinal fluid, pleural fluid, ascites fluid, sputum, stool, or biopsy sample.
  • the nucleic acid-containing sample used for detection of methylated CpG may be from any source including, but not limited to, brain, colon, urogenital, hematopoietic, thymus, testis, ovarian, uterine, prostate, breast, colon, lung and renal tissue and may be extracted by a variety of techniques such as that described by Maniatis, et al. ⁇ Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY, pp 280, 281, 1982).
  • the nucleic acid of interest can be any nucleic acid where it is desirable to detect the presence of a CpG island.
  • the CpG island comprises a CpG island located in a gene.
  • a "CpG island” is a CpG rich region of a nucleic acid sequence.
  • the nucleic acid sequence may be, for example, a pl6, a Rb, a VHL, a hMLHI, or a BRCAl gene.
  • the nucleic acid of interest can be, for example, a MINTl -33 nucleic acid sequence.
  • any gene or nucleic acid sequence of interest containing a CpG sequence can be detected using the method of the invention.
  • the presence of methylated CpG in the nucleic acid-containing specimen may be indicative of a disorder.
  • the disorder is a cell proliferative disorder.
  • a "cell proliferative disorder” is any disorder in which the proliferative capabilities of the affected cells is different from the normal proliferative capabilities of unaffected cells.
  • An example of a cell proliferative disorder is neoplasia. Malignant cells (i.e., cancer) develop as a result of a multistep process.
  • disorders associated with increased methylation of CpG-islands are colon cancer, lung cancer, renal cancer, leukemia, breast cancer, prostate cancer, uterine cancer, astrocytoma, glioblastoma, and neuroblastoma.
  • the disorder is an age-associated disorder.
  • age-associated disorder is used to describe a disorder observed with the biological progression of events occurring over time in a subject.
  • the subject is a human.
  • Non-limiting examples of age-associated disorders include, but are not limited to, atherosclerosis, diabetes melitis, and dementia.
  • An age-associated disorder may also be a cell proliferative disorder. Examples of age-associated disorders which are cell proliferative disorders include colon cancer, lung cancer, breast cancer, prostate cancer, and melanoma, amongst others.
  • An age-associated disorder is further intended to mean the biological progression of events that occur during a disease process that affects the body, which mimic or substantially mimic all or part of the aging events which occur in a normal subject, but which occur in the diseased state over a shorter period of time.
  • the age-associated disorder is a "memory disorders or learning disorders" which are characterized by a statistically significant decrease in memory or learning assessed over time by the Randt Memory Test (Randt et al, Clin. Neuropsychol. , 2:184, 1980), Wechsler Memory Scale (J Psych., 12:87-95, 1945), Forward Digit Span test (Craik, Age Differences in Human Memory, in: Handbook of the Psychology of Aging. Birren, J., and Schaie, K., Eds., New York, Van Nostrand, 1977), Mini-Mental State Exam (Folstein et al, J. of Psych. Res.
  • the sample may be treated before amplification with a reagent effective for opening the cells, fluids, tissues, or animal cell membranes of the sample, and for exposing the nucleic acid(s).
  • a reagent effective for opening the cells, fluids, tissues, or animal cell membranes of the sample, and for exposing the nucleic acid(s).
  • a method for identifying a methylated
  • CpG-containing nucleic acid including contacting a nucleic acid sample suspected of comprising a CpG-containing nucleic acid with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites under conditions and for a time to allow cleavage of unmethylated nucleic acid.
  • the sample is further contacted with an isoschizomer of the methylation sensitive restriction endonuclease, that cleaves both methylated and unmethylated CpG-sites, under conditions and for a time to allow cleavage of methylated nucleic acid.
  • Oligonucleotides are added to the nucleic acid sample under conditions and for a time to allow ligation of the oligonucleotides to nucleic acid cleaved by the restriction endonuclease, and the digested nucleic acid is amplified. Following identification, the methylated CpG-containing nucleic acid can be cloned, using method well known to one of skill in the art (see Sambrook et al. , Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Press, 1989).
  • a "methylation sensitive restriction endonuclease” is a restriction endonuclease that includes CG as part of its recognition site and has altered activity when the C is methylated as compared to when the C is not methylated.
  • the methylation sensitive restriction endonuclease has inhibited activity when the C is methylated (e.g., Smal).
  • Specific non-limiting examples of a methylation sensitive restriction endonucleases include Smal, BssHll, or Hpall. Such enzymes can be used alone or in combination.
  • methylation sensitive restriction endonucleases will be known to those of skill in the art and include, but are not limited to S ⁇ cII, Eagl, and BstUl, for example.
  • An "isoschizomer" of a methylation sensitive restriction endonuclease is a restriction endonuclease which recognizes the same recognition site as a methylation sensitive restriction endonuclease but which cleaves both methylated and unmethylated CGs.
  • One of skill in the art can readily determine appropriate conditions for a restriction endonuclease to cleave a nucleic acid (see Sambrook et al, Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Press, 1989). Without being bound by theory, actively transcribed genes generally contain fewer methylated CGs than in other genes.
  • a nucleic acid of interest is cleaved with a methylation sensitive endonuclease.
  • cleavage with the methylation sensitive endonuclease creates a sufficient overhang on the nucleic acid of interest.
  • the cleavage product can still have a sufficient overhang.
  • An "overhang” refers to nucleic acid having two strands wherein the strands end in such a manner that a few bases of one strand are not base paired to the other strand.
  • a "sufficient overhang” refers to an overhang of sufficient length to allow specific hybridization of an oligonucleotide of interest.
  • a sufficient overhang is at least two bases in length. In another embodiment, the sufficient overhang is four or more bases in length.
  • An overhang of a specific sequence on the nucleic acid of interest may be desired in order for an oligonucleotide of interest to hybridize. In this case, the isoschizomer can be used to create the overhang having the desired sequence on the nucleic acid of interest.
  • the cleavage with a methylation sensitive endonuclease results in a reaction product of the nucleic acid of interest that has a blunt end or an insufficient overhang.
  • an isoschizomer of the methylation sensitive restriction endonuclease can create a sufficient overhang on the nucleic acid of interest.
  • Boilt ends refers to a flush ending of two stands, the sense stand and the antisense strand, of a nucleic acid.
  • an oligonucleotide is ligated to the nucleic acid cleaved of interest which has been cleaved by the methylation specific restriction endonuclease.
  • “Ligation” is the attachment of two nucleic acid sequences by base pairing of substantially complementary sequences or by the formation of a covalent bonds between two nucleic acid sequences.
  • An "oligonucleotide” is a nucleic acid sequence of 2 to 40 bases in length. Preferably the oligonucleotide is from 15 to 35 bases in length. In one embodiment, the oligonucleotide is ligated to the overhang on the nucleic acid sequence of interest by base pairing.
  • two oligonucleotides are utilized to form an adaptor.
  • An "adaptor" is a double-stranded nucleic acid sequence with one end that has a sufficient single-stranded overhang at one or both ends such that the adaptor can be ligated by base-pairing to a sufficient overhang on a nucleic acid of interest that has been cleaved by a methylation sensitive restriction enzyme or an isoschizomer of a methylation sensitive restriction enzyme.
  • two oligonucleotides can be used to form an adaptor; these oligonucleotides are substantially complementary over their entire sequence except for the region(s) at the 5' and/or 3' ends that will form a single stranded overhang.
  • the single stranded overhang is complementary to an overhang on the nucleic acid cleaved by a methylation sensitive restriction enzyme or an isoschizomer of a methylation sensitive restriction enzyme, such that the overhang on the nucleic acid of interest will base pair with the 3 ' or 5' single stranded end of the adaptor under appropriate conditions.
  • the conditions will vary depending on the sequence composition (GC vs AT), the length, and the type of nucleic acid (see Sambrook et al, Molecular Cloning: a Laboratory Manual. 2nd Ed.; Cold Spring Harbor Laboratory Press, Plainview, NY, 1998).
  • the nucleic acid of interest is amplified using a primer complementary to the oligonucleotide.
  • primer refers to a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and most preferably more than 8, which sequence is capable of initiating synthesis of a primer extension product, which is substantially complementary to a nucleic acid such as an adaptor or a ligated oligonucleotide.
  • Environmental conditions conducive to synthesis include the presence of nucleoside triphosphates and an agent for polymerization, such as DNA polymerase, and a suitable temperature and pH.
  • the primer is preferably single stranded for maximum efficiency in amplification, but may be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligodeoxyribo-nucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent for polymerization. The exact length of primer will depend on many factors, including temperature, buffer, and nucleotide composition.
  • the oligonucleotide primer typically contains 12-20 or more nucleotides, although it may contain fewer nucleotides.
  • Primers of the invention are designed to be “substantially” complementary to each strand of the oligonucleotide to be amplified and include the appropriate G or C nucleotides as discussed above. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions which allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with a 5' and 3' oligonucleotide to hybridize therewith and permit amplification of CpG containing nucleic acid sequence.
  • Primers of the invention are employed in the amplification process which is an enzymatic chain reaction that produces exponential quantities of target locus relative to the number of reaction steps involved (e.g., polymerase chain reaction or PCR).
  • one primer is complementary to the negative (-) strand of the locus and the other is complementary to the positive (+) strand.
  • Annealing the primers to denatured nucleic acid followed by extension with an enzyme, such as the large fragment of DNA Polymerase I (Klenow) and nucleotides results in newly synthesized + and - strands containing the target locus sequence.
  • the product of the chain reaction is a discrete nucleic acid duplex with termini corresponding to the ends of the specific primers employed.
  • oligonucleotide primers of the invention may be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof.
  • diethylphosphoramidites are used as starting materials and may be synthesized as described by Beaucage, et al. (Tetrahedron Letters, 22: 1859-1862, 1981).
  • Beaucage, et al. Tetrahedron Letters, 22: 1859-1862, 1981.
  • One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066.
  • Strand separation can be effected either as a separate step or simultaneously with the synthesis of the primer extension products. This strand separation can be accomplished using various suitable denaturing conditions, including physical, chemical, or enzymatic means, the word "denaturing" includes all such means.
  • One physical method of separating nucleic acid strands involves heating the nucleic acid until it is denatured. Typical heat denaturation may involve temperatures ranging from about 80° to 105°C for times ranging from about 1 to 10 minutes.
  • Strand separation may also be induced by an enzyme from the class of enzymes known as helicases or by the enzyme RecA, which has helicase activity, and in the presence of riboATP, is known to denature DNA.
  • an enzyme from the class of enzymes known as helicases or by the enzyme RecA which has helicase activity, and in the presence of riboATP, is known to denature DNA.
  • the reaction conditions suitable for strand separation of nucleic acids with helicases are described by Kuhn Hoffmann-Berling (CSH-Quantitative Biology, 43:63, 1978) and techniques for using RecA are reviewed in C. Radding (Ann. Rev. Genetics, 16:405-437, 1982).
  • the separated strands are ready to be used as a template for the synthesis of additional nucleic acid strands.
  • This synthesis is performed under conditions allowing hybridization of primers to templates to occur. Generally synthesis occurs in a buffered aqueous solution, generally at a pH of about 7- 9. Preferably, a molar excess (for genomic nucleic acid, usually about 10 8 :1 prime ⁇ template) of the two oligonucleotide primers is added to the buffer containing the separated template strands.
  • the amount of complementary strand may not be known if the process of the invention is used for diagnostic applications, so that the amount of primer relative to the amount of complementary strand cannot be determined with certainty.
  • the amount of primer added will generally be in molar excess over the amount of complementary strand (template) when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands. A large molar excess is preferred to improve the efficiency of the process.
  • the deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to about 90°- 100°C from about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period, the solution is allowed to cool to approximately room temperature, which is preferable for the primer hybridization. To the cooled mixture is added an appropriate agent for effecting the primer extension reaction (called herein "agent for polymerization”), and the reaction is allowed to occur under conditions known in the art.
  • agent for polymerization may also be added together with the other reagents if it is heat stable.
  • This synthesis (or amplification) reaction may occur at room temperature up to a temperature above which the agent for polymerization no longer functions.
  • the temperature is generally no greater than about 40°C. Most conveniently the reaction occurs at room temperature.
  • the agent for polymerization may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes.
  • Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, polymerase muteins, reverse transcriptase, and other enzymes, including heat-stable enzymes (i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation).
  • Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each locus nucleic acid strand.
  • the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths.
  • agents for polymerization may be agents for polymerization, however, which initiate synthesis at the 5' end and proceed in the other direction, using the same process as described above.
  • the method of amplifying is by PCR, as described herein and as is commonly used by those of ordinary skill in the art. However, alternative methods of amplification have been described and can also be employed.
  • the nucleic acid can be attached to a solid support, such as a membrane, and can be hybridized with any probe of interest, to detect any nucleic acid sequence.
  • a solid support such as a membrane
  • membranes are known to one of skill in the art for the adhesion of nucleic acid sequences. Specific non- limiting examples of these membranes include nitrocellulose (Nitropure) or other membranes used in for detection of gene expression such as polyvinylchloride, diazotized paper and other commercially available membranes such as GenescreenTM, ZetaprobeTM (Biorad), and NytranTM. Methods for attaching nucleic acids to these membranes are well known to one of skill in the art. Alternatively, screening can be done in a liquid phase.
  • nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
  • An example of progressively higher stringency conditions is as follows: 2 x SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68°C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each ofthe conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all ofthe steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically. In general, conditions of high stringency are used for the hybridization ofthe probe of interest.
  • the probe of interest can be detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme.
  • a radioisotope for example, with a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme.
  • Those of ordinary skill in the art will know of other suitable labels for binding to the probe, or will be able to ascertain such, using routine experimentation.
  • representational difference analysis can be performed on CpG-containing nucleic acid following MCA.
  • MCA utilizes kinetic and subtractive enrichment to purify restriction endonuclease fragments present in one population of nucleic acid fragments but not in another.
  • RDA enables the identification of small differences between the sequences of two nucleic acid populations.
  • RDA uses nucleic acid from one population as a "tester” and nucleic acid from a second population as a "driver,” in order to clone probes for single copy sequences present in (or absent from) one ofthe two populations.
  • nucleic acid from a "normal” individual or sample, not having a disorder such as a cell- proliferative disorder is used as a "driver”
  • nucleic acid from an "affected” individual or sample, having the disorder such as a cell proliferative disorder is used as a "tester.”
  • the nucleic acid used as a "tester” is isolated from an individual having a cell proliferative disorder such as colon cancer, lung cancer, renal cancer, leukemia, breast cancer, prostate cancer, uterine cancer, astrocytoma, glioblastoma, and neuroblastoma.
  • the nucleic acid used as a "driver” is thus normal colon, normal lung, normal kidney, normal blood cells, normal breast, normal prostate, normal uterus, normal astrocytes, normal glial and normal neurons, respectively.
  • the nucleic acid used as a "driver” is isolated from an individual having a cell proliferative disorder such as colon cancer, lung cancer, renal cancer, leukemia, breast cancer, prostate cancer, uterine cancer, astrocytoma, glioblastoma, and neuroblastoma.
  • the nucleic acid used as a "tester” is thus normal colon, normal lung, normal kidney, normal blood cells, normal breast, normal prostate, normal uterus, normal astrocytes, normal glial and normal neurons, respectively.
  • One of skill in the art will readily be able to identify the "tester" nucleic acid useful with to identify methylated nucleic acid sequences in given "driver" population.
  • the invention provides a method for identifying an agent which can affect methylation.
  • An agent can affect methylation by either increasing or decreasing methylation.
  • the method includes incubating an agent and a sample containing a CpG-containing polynucleotide under conditions sufficient to allow the components to interact, and measuring the effect ofthe compound on the methylation ofthe CpG-containing nucleic acid.
  • the sample is a cell expressing a polynucleotide of interest.
  • the sample is substantially purified nucleic acid.
  • Substantially purified nucleic acid is nucleic acid which has been separated from the cellular components which naturally accompany it, or from contaminating elements such as proteins, lipids, or chemical resins.
  • Substantially pure nucleic acid can be extracted from any cell type, or can be chemically synthesized. Purity can be measured by any appropriate method, such as measuring the absorbance of light (e.g., A 26 o A 280 ratio).
  • the nucleic acid can be identified by the methylated CpG island amplification, as described above.
  • the methylation ofthe polynucleotide in the sample can then be compared to the methylation of a control sample not incubated with the agent.
  • the effect ofthe agent on methylation of a polynucleotide can be measured by assessing the methylation ofthe polynucleotide by the methods ofthe invention.
  • the effect of the agent on methylation of a polynucleotide can be measured by assessing the expression ofthe polynucleotide of interest. Means of measuring expression are well known to one of skill in the art (e.g., Northern blotting or RNA dot blotting, amongst others).
  • the agents which affect methylation can include peptides, peptidomimetics, polypeptides, pharmaceuticals, and chemical compounds and biological agents. Psychotropic, antiviral, and chemotherapeutic compounds can also be tested using the method ofthe
  • “Incubating” includes conditions which allow contact between the test agent and the cell of interest.
  • Contacting includes in solution and solid phase.
  • the test agent may also be a combinatorial library for screening a plurality of compounds.
  • Agents identified in the method ofthe invention can be further cloned, sequenced, and the like, either in solution of after binding to a solid support, by any method usually applied to the isolation of a specific DNA sequence Molecular techniques for DNA analysis (Landegren et al. , Science 242:229-237. 1988) and cloning have been reviewed (Sambrook et al, Molecular Cloning: a Laboratory Manual. 2nd Ed.; Cold Spring Harbor Laboratory Press, Plainview, NY, 1998.
  • the sample can be any sample of interest.
  • the sample may be a cell sample or a membrane sample prepared from a cell sample.
  • Suitable cells include any host cells containing a nucleic acid including a CpG island.
  • the cells can be primary cells or cells of a cell line.
  • the agent is incubated with the sample of interest suspected of including a CpG-containing nucleic acid and methylation is evaluated by MCA.
  • nucleic acid from the sample suspected of including a CpG-containing nucleic acid is contacted with a methylation sensitive restriction endonuclease which cleaves only unmethylated CpG sites under conditions and for a time to allow cleavage of unmethylated nucleic acid.
  • An isoschizomer ofthe methylation sensitive restriction endonuclease is also utilized.
  • An oligonucleotide is added to the nucleic acid sample under conditions and for a time to allow ligation ofthe oligonucleotide to nucleic acid cleaved by said restriction endonuclease, and the digested nucleic acid is amplified.
  • the digested nucleic acid is adhered to a membrane, and the membrane is hybridized with a probe of interest.
  • representation difference analysis can also be performed.
  • kits may comprise a carrier means containing one or more container means such as vials, tubes, and the like, each ofthe container means comprising one ofthe separate elements to be used in the method.
  • One ofthe container means can comprise a container containing an oligonucleotide for ligation to nucleic acid cleaved by a methylation sensitive restriction endonuclease.
  • One or more container means can also be included comprising a primer complementary to the oligonucleotide.
  • one or more container means can also be included which comprise a methylation sensitive restriction endonuclease.
  • One or more container means can also be included containing an isoschizomer of said methylation sensitive restriction enzyme.
  • the kit may comprise a carrier means containing one or more container means comprising a solid support, wherein the solid support has a nucleic acid sequence selected from the group consisting of MINT 1-33 immobilized on the solid support.
  • the solid support is a membrane.
  • membranes are known to one of skill in the art for the adhesion of nucleic acid sequences. Specific non-limiting examples of these membranes include nitrocellulose (Nitropure) or other membranes used in for detection of gene expression such as polyvinylchloride, diazotized paper and other commercially available membranes such as GenescreenTM, ZetaprobeTM (Biorad), and NytranTM.
  • the MINTl -33 sequences immobilized on the solid support can then be hybridized to nucleic acid sequences produced by performing the MCA procedure on the nucleic acids of a sample of interest in order to determine if the nucleic acid sequences contained in the sample are methylated.
  • the invention provides isolated MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polynucleotides ( SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively).
  • polynucleotides include DNA, cD A and RNA sequences which encode MINTl , MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINTl 5, MINTl 7, MINT19, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polypeptides. It is understood that naturally occurring, synthetic, and intentionally manipulated polynucleotides are included.
  • MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINT15, MINT17, MINT 19, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 nucleic acids may be subjected to site-directed mutagenesis.
  • the nucleic acid sequence for MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 also includes antisense sequences, and sequences encoding dominant negative forms of MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MONTI 0, MINT14, MINT 15, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31 , MINT32 and MINT33.
  • the invention provides methylated and unmethylated forms of MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polynucleotides ( SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,
  • Methylated nucleic acid sequences are also provided which include MINT3, MINT5, MINT 7, MINTl 1, MINT12, MINTl 3, MINT16, MINT18, MINT21, MINT25, MINT26, MINT28, and MINT29 (SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29, respectively). It is understood that naturally occurring, synthetic, and intentionally manipulated polynucleotides are included.
  • the polynucleotides ofthe invention includes "degenerate variants" sequences that are degenerate as a result ofthe genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of a polypeptide encoded by the nucleotide sequence of SEQ ID NOs: 1-33 is functionally unchanged.
  • MINTl methylated and unmethylated isolated polynucleotide sequences of MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33.
  • the nucleotide sequence is SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively.
  • the nucleotide sequence is SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29, respectively.
  • polynucleotide or “nucleic acid sequence” refers to a polymeric form of nucleotides at least 10 bases in length.
  • isolated polynucleotide is meant a polynucleotide that is not immediately contiguous with both ofthe coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences.
  • the nucleotides ofthe invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.
  • the term includes single and double forms of DNA.
  • the polynucleotide encoding MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 includes SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, dominant negative forms of MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINTl 5,
  • a complementary sequence may include an antisense nucleotide.
  • the sequence is RNA
  • the deoxynucleotides A, G, C, and T of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 are replaced by ribonucleotides A, G, C, and U, respectively.
  • fragments ofthe above-described nucleic acid sequences that are and are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA that encoded by SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 under physiological conditions or a close family member of MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30,
  • MINT31, MINT32 and MINT33 The term “selectively hybridize” refers to hybridization under moderately or highly stringent conditions which excludes non-related nucleotide sequences. Hybridization conditions have been described above.
  • the MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINT15, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32, and MINT33 nucleotide sequence includes the disclosed sequence and conservative variations ofthe polypeptides encoded by MINT 1 , MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polynucleotides.
  • conservative variation denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like.
  • conservative variation also includes the use of a substituted arnino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
  • MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINT15, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 nucleic acid sequences can be expressed in vitro by DNA transfer into a suitable host cell.
  • "Host cells” are cells in which a vector can be propagated and its DNA expressed.
  • the cell may be prokaryotic or eukaryotic.
  • the term also includes any progeny ofthe subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
  • the MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31 , MINT32 and MINT33 polynucleotide sequences may be inserted into an expression vector.
  • expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation ofthe sequence of interest genetic sequences. Polynucleotide sequence which encode sequence of interest can be operatively linked to expression control sequences.
  • “Operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • An expression control sequence operatively linked to a coding sequence is ligated such that expression ofthe coding sequence is achieved under conditions compatible with the expression control sequences.
  • expression control sequences refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation ofthe nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • control sequences is intended to included, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • Expression control sequences can include a promoter.
  • promoter is meant minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions ofthe gene. Both constitutive and inducible promoters, are included in the invention (see, e.g., Bitter et al, Methods in Enzymology 1£3_:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
  • promoters derived from the genome of mammalian cells e.g., metallothionein promoter
  • mammalian viruses e.g., the retro virus long terminal repeat; the adeno virus late promoter; the vaccinia virus 7.5K promoter
  • Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription ofthe nucleic acid sequences ofthe invention.
  • the MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINTl 5, MINTl 7, MINT19, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polynucleotide sequence may be inserted into an expression vector which contains a promoter sequence which facilitates the efficient transcription ofthe inserted genetic sequence ofthe host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection ofthe transformed cells.
  • Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg et al.
  • the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g. , T7, metallothionein I, or polyhedron promoters).
  • a promoter e.g. , T7, metallothionein I, or polyhedron promoters.
  • MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINT15, MINTl 7, MINT19, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polynucleotide sequences can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences ofthe invention.
  • transformation is meant a genetic change induced in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell).
  • new DNA i.e., DNA exogenous to the cell.
  • the genetic change is generally achieved by introduction ofthe DNA into the genome ofthe cell (i.e., stable).
  • transformed cell is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding sequence of interest. Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method using procedures well known in the art. Alternatively, MgCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast ofthe host cell if desired.
  • Eukaryotic cells can also be cotransformed with DNA sequences encoding the sequence of interest, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors. Cold Spring Harbor Laboratory, Gluzman ed., 1982).
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • Isolation and purification of microbial expressed polypeptide, or fragments thereof, provided by the invention may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
  • the invention provides substantially purified polypeptide encoded by MINTl, MINT2, MINT4, MINT6.
  • MINT8 MINT 9, MINT10, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polynucleotide sequences.
  • substantially purified refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify a polypeptide encoded by MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31 , MINT32 and MINT33 polynucleotide sequence using standard techniques for protein purification.
  • the substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel.
  • MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31 , MINT32 and MINT33 polypeptide can also be determined by amino-terminal amino acid sequence analysis.
  • polypeptides ofthe invention also include dominant negative forms of the MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 or MINT33 polypeptide which do not have the biological activity of MINTl , MINT2, MINT4,
  • MINT27, MINT30, MINT31, MINT32, or MINT33 is a polypeptide that is structurally similar to MINTl, MTNT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 or MINT33 polypeptide but does not have wild-type MINTl , MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 or MINT33 function.
  • a dominant-negative MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 or MINT33 polypeptide may interfere with wild-type MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24,
  • MINT27, MINT30, MINT31, MINT32 or MINT33 function by binding to, or otherwise sequestering, regulating agents, such as upstream or downstream components, that normally interact functionally with the MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 or MINT33 polypeptide.
  • the principle underlying MCA involves amplification of closely spaced methylated Smal sites to enrich for methylated CGIs.
  • the MCA technique is outlined in Figure 1A. About 70 to 80% of CpG islands contain at least two closely spaced ( ⁇ lkb) Smal sites (CCCGGG). Only those Smal sites within these short distances can be amplified using MCA, ensuring representation ofthe most CpG rich sequences. Briefly, DNA is digested with Smal, which cleaves only unmethylated sites, leaving blunt ends between the C and G. DNA is then digested with the Smal isoschizomer Xmal, which does cleave methylated CCCGGG sites, and which leaves a 4 base overhang. Adaptors are ligated to this overhang, and PCR is performed using primers complementary to these adaptors. The amplified DNA is then spotted on a nylon membrane and can be hybridized with any probe of interest.
  • MCA DNA from Caco2 and normal colon mucosa were mixed in various proportions, and the methylation level of each mix was determined using MCA.
  • MCA detected pi 6 methylation in a semi- quantitative manner between 1% and 100% methylated alleles.
  • MCA was performed on 109 samples of normal colonic mucosa and adjacent primary colorectal tumor that had previously been typed for pi 6 methylation by Southern blot analysis (Ahuja et al, 1997). MCA and Southern blot were concordant in 107/109 (98%) ofthe cases. In one case, MCA detected a low level of methylation (5-10 %) in a cancer sample that had been judged negative by Southern blot.
  • MCA is a novel PCR-based technique that allows for the rapid enrichment of hypermethylated CG rich sequences, with a high representation of methylated CpG islands. This technique can have several potential applications. MCA is very useful for the determination ofthe methylation status of a large number of samples at multiple loci simultaneously. By optimizing the PCR conditions, it should be readily adaptable to the study of the methylation status of any gene that has two closely spaced Smal sites.
  • MCA there is a very high concordance rate between MCA and other methods for the detection of hypermethylation such as Southern blot analysis and bisulfite-based methods.
  • MCA (1) requires good quality DNA, excluding the study of paraffin-embedded samples, (2) examines only a limited number of CpG sites within a CGI and (3) is sensitive to incomplete digestion using the methylation-sensitive enzyme Smal. Nevertheless, many steps in MCA are amenable to automation and, by allowing for the examination of multiple genes relatively quickly, may have important applications in population-based studies of CGI methylation.
  • promoter prediction was performed using several computer programs (see programs available at http://dot.imgen.bcm.tmc.edu:9331/seq-search/gene-search.html). Twenty out ofthe 33 clones were predicted as promoters using the NNPP program, and 6 were predicted as promoters by using the TSSG program.
  • the chromosomal position of most ofthe unknown clones was determined using a somatic cell hybrid panel and a radiation hybrid panel (Table 1).
  • MINT3 and MINT9 mapped to chromosome lp35-36
  • MINT 13 mapped to 7q31
  • MINT24 mapped to 3p25-26
  • MINT25 mapped to 22ql 1-Ter
  • MINT31 mapped to 17q21. All of these chromosomal segments are areas that are frequently deleted in various tumors.
  • MCA myelogenous coactivation protein
  • RDA Reactive adenosine deficiency protein
  • MCA coupled with RDA is a rapid and powerful technology for this purpose, and compares favorably with other described techniques (Hayashizaki et al, 1994, Gonzalgo et al, 1997; Huang et al, 1997).
  • MCA could potentially be used to discover novel imprinted genes using parthenogenetic DNA (Kaneko-Ishino et al. , 1995), as well as novel X-chromosome genes.
  • Versican is a secreted glycoprotein that appears to be regulated by the Rb tumor suppressor gene (Rohde et al, 1996).
  • MINTl 1 corresponds to part of exon 1 and part of intron 1 ofthe versican gene ( Figure 3 A). Hypermethylation ofthe two Smal sites in exon 1 and intron 1 in colon cancer cell lines was confirmed by both Southern blot analysis and MCA. In order to determine if this methylation was representative ofthe entire CGI, including the proximal promoter, PCR was performed on bisulfite-treated DNA using primers designed to amplify the region around the transcription start site of this gene.
  • the PCR product was then digested with restriction enzymes that distinguish methylated from unmethylated DNA.
  • the versican promoter was found to be completely methylated in the colon cancer cell lines, DLD1, LOVO, SW48 and SW837, and partially methylated in HCT116 and HT29 (Figure 3B).
  • versican was hypermethylated in 17 out of 25 cases (68%).
  • some methylation ofthe versican promoter was also found in normal tissues, albeit at lower levels when compared to tumors.
  • the level of methylation in normal colon mucosa increased with age ofthe patient (Fig3C), from an average of 6.9% in patients between 20 and 30 years of age, to an average of 28.9% in patients over 80.
  • methylation ofthe alpha-tubulin gene in Caco2 is consistent with the results of studying the gene expression profile of colorectal cancers using SAGE (Zang et al, 1997), which demonstrated that alpha-tubulin is markedly down-regulated in CRC.
  • Methylation ofthe CSX and OPT genes does not coincide with their 5' end, and is therefore not expected to silence these genes. It is possible, however, that these CpG islands are associated with alternate transcripts ofthe genes, or with other nearby genes, which would then be silenced by methylation (Wutz, A., et al, 1997).
  • methylation of ribosomal genes has previously been seen in aging tissues (Swisshelm, K., et al, 1990) and therefore is not surprising to find in cancers. Because some of the clones recovered are in the exon 1 region of expressed genes, identification of new tumor suppressor genes might be facilitated by using MCA RDA clones as probes for screening cDNA library. Indeed, based on their chromosome location, several clones map to chromosomal regions thought to harbor TSGs because they are highly deleted in various tumors (e.g. , chromosome lp35, 3p25-26, 7q31, 17q21 and 22ql l-Ter).
  • the remaining 7 clones were methylated exclusively in CRC, and their frequency of methylation was significantly lower than type A methylation (ranging from 10% to 50%). This type of methylation was named type C for cancer-specific.
  • Type A methylation was seen in the majority of these clones: 22 of 29 (74%) clones were methylated in an age-related manner in normal colon tissue, and hypermethylated at a high frequency in CRC, as we have shown for the ER gene (Issa et al, 1994) and others (Issa et al, 1996; Ahuja et al. , submitted). These results suggest that a large number of CGIs in the human genome are incrementally methylated during the aging process and, for many genes, this methylation correlates with reduced gene expression as shown for ER (Issa et al. , 1994) and versican.
  • Type A methylation Although the mechanism of Type A methylation is unknown, it is likely to result from physiological processes rather than a genetic alteration because (1) it is very frequent and affects large numbers of cells, (2) it is present in all individuals, not just patients with cancer and (3) this process is gene and tissue specific (Ahuja et al, submitted).
  • methylation status at a given CGI is thought to be related to positive (methylator) factors (Mummaneni et al, 1993; Mummaneni et al, 1995; Magewu and Jones, 1994, Nertino et al, 1996) and negative (protector) factors (Macleod et al, 1994; Brandeis et al, 1994; Turker and Bestor, 1997; Chen et al, 1997), it is possible that for some genes, this balance favors slightly de-novo methylation, and that this is reflected by progressive hypermethylation after repeated cell divisions.
  • the 50 CRC fell into two distinct groups: (1) A group with a high level of Type C methylation, whereby all the tumors had methylation of 4 or more loci simultaneously and (2) a group where methylation of any type C clone is extremely rare.
  • the first group of tumors appears to display profound global hypermethylation (GH+), which is lacking in the second group (GH-).
  • GH+ global hypermethylation
  • GH- GH-
  • Type A methylation was not significantly different between GH+ and GH- tumors (Table 2).
  • GH was also detected in a subset of colorectal adenomas (Figure 5), suggesting that it is an early event in carcinogenesis.
  • 5 of 5 small adenomas ( ⁇ 7 mm) were GH-
  • 6 of 9 large adenomas (>10 mm) were GH+, suggesting that this defect may be acquired in the transition between small and large adenomas.
  • both an adenoma and a cancer from the same patients were examined. In one of these, GH was detected both in the adenoma and the cancer; in 3 cases, GH was detected in the cancer but not in the adenoma and in 2 cases, GH was detected in neither the adenoma nor the cancer.
  • type C methylation is relatively infrequent in primary CRC, and is never observed in normal colon mucosa. Furthermore, detailed analysis of type C methylation in CRC revealed a striking pattern, suggesting the presence of global hypermethylation in a subset of these tumors: GH positive cases are characterized by frequent and concordant methylation of all type C clones examined, such that each tumor has at least four methylation events. By contrast, type C methylation is virtually non-existent in tumors without GH.
  • type C methylation an epigenetic error
  • this epigenetic defect may then result in additional genetic lesions through the induction of mismatch-repair deficiency.
  • hMLHI was studied by bisulfite-PCR only because it does not have 2 Smal sites in its CGI. Overall, 16 out of 50 (32%) cancers had evidence of microsatellite instability. Among the 29 GH+ cases, 12 had evidence of hMLHI methylation, suggesting that hMLHI is one ofthe targets of global hypermethylation in CRC. All of these 12 tumors had microsatellite instability. By contrast, hMLHI methylation was detected in only one ofthe 21 GH- cases. These data establish a strong link between the GH phenotype, hMLHI methylation and microsatellite instability in CRC.
  • GH is detectable in about half of colonic adenomas, but none of these tumors have hMLHI methylation, and microsatellite instability is extremely rare in this pre- neoplastic lesion (Samowitz and Slattery, 1997).
  • CGI methylation plays two distinct roles, and appears to arise through distinct mechanisms.
  • type A methylation arises as a function of age in normal colorectal epithelial cells.
  • genes that regulate the growth and/or differentiation of these cells such methylation results in a hyperproliferative state, which is thought to precede tumor formation in the colon (reviewed by Lipkin, 1988).
  • hyperproliferation is known to arise with age in colorectal epithelium (Holt et al, 1988, Roncucci et al, 1988), and to be marked in patients with CRC.
  • type A methylation The cause of type A methylation is unknown, but without being bound by theory it is possible that it is related to endogenous factors inherent to the structure of DNA, and that it may be modulated by factors such as level of ongoing expression and exposure to carcinogenic insults. Furthermore, modulation of type A methylation may provide one possible explanation for the reduction in CRC tumorigenesis by reducing levels of DNA-methyltransferase (Laird et al, 1995). A second major role for CGI methylation appears later, perhaps at the transition between small and large adenomas in the colon. This methylation (type C) affects only a subset of tumors, which then evolve along a pathway of global hypermethylation.
  • This GH leads to cancer development through the simultaneous inactivation of multiple tumor-suppressor genes such as p 16, and induction of mismatch repair deficiency through inactivation of hMLHI .
  • the cause of this global hypermethylation is unknown, but may well be related to inactivation of a gene that protects CGIs from de-novo methylation.
  • tumors without GH evolve along more classic genetic instability pathways, including chromosomal instability (Lengauer et al. , 1997 A).
  • Lengauer et al found an inverse co ⁇ elation between chromosomal instability and MMR deficiency in CRC cell lines (Lengauer et al, 1997B).
  • MCA has been developed to selectively amplify methylated CGIs.
  • MCA/RDA 33 differentially methylated clones in CRC were isolated.
  • the methylation profile of these clones revealed that nearly all methylation in CRC can be accounted for by (1) age-related methylation and (2) a hypermethylator phenotype presumably caused by global hypermethylation. Deciphering the mechanisms underlying these phenomena should facilitate the early detection, prevention and therapy of cancers, including colorectal cancers.
  • methylated CpG island amplification was used followed by representational difference analysis (Razin and Cedar, Cell 17: 473-476, 1994, herein incorporated by reference).
  • MINT31 representational difference analysis
  • a Blast search revealed this fragment to be completely identical to part of a BAC clone (Genbank: AC004590) sequenced by high throughput genomic sequence.
  • the region su ⁇ ounding MINT31 fulfills the criteria of a CpG island: GC content 0.67, CpG/GpC ratio 0.78 and a total of 305 CpG sites in a 4 kb region.
  • the MINT31 CpG island corresponds to the 3' regions of Gl and G2, based on the direction of the open reading frame and the presence of a poly A tail, and is unlikely to influence their transcription.
  • the EST closest to MINT31 (HI 3333) was sequenced entirely and was found not to contain a continuous open reading frame, but a poly-adenylation signal was identified on one end, along with a poly A tail.
  • the MINT31 CpG island is also in the 5' region of human CACNAIG gene and may play a role in its transcriptional activity.
  • the human CACNAIG sequence deposited in Genbank lacks the 5' region ofthe gene, when compared to the rat homologue.
  • To determine the 5' region of human CACNAIG we amplified cDNA by RT-PCR using primers based on the BAC sequence (Genbank: AC004590, herein incorporated by reference). The PCR products were cloned and sequenced, and the genomic organization ofthe gene was determined by comparing the newly identified sequences as well as the known sequences to the BAC that covers this region.
  • CACNAIG is composed of 34 exons which span a 70 kb area. Based on sequences deposited in Genbank, the gene has two possible 3' ends caused by alternate splicing.
  • CACNAIG is highly homologous to rat CACNAIG with 93% identity at the protein level, and 89% identity at the nucleotide level.
  • the 5' flanking region of CACNAIG lacks TATA and CAAT boxes, which is similar to many housekeeping genes.
  • a putative TFIID binding site was identified 547-556 bp upstream from the translation start site, and several other potential transcription factor binding sites such as API (1 site), AP2 (2 sites) and SP1 (10 sites), were identified upstream of CACNAIG exon 1 using the promoter prediction program, TESS (data not shown).
  • the CACNAIG CpG island is 4 kb, and is larger than many typical CpG islands. MINT31 corresponds to the 5' edge ofthe island while CACNA1G is in the 3' region. It is not known whether large CpG islands such as this are coordinately regulated with regards to protection from methylation, and aberrant methylation in cancer. To address this issue, the methylation status ofthe 5' region of CACNAIG was studied using bisulfite-PCR of DNA from normal tissues as well as 35 human cancer cell lines from colon, lung, prostate, breast and hematopoietic tumors. The CpG island was divided into 8 regions and their methylation status was examined separately.
  • the genomic DNA was treated with sodium bisulfite and PCR amplified using primers containing no or a minimum number of CpG sites. Methylated alleles were detected by digesting the PCR products using restriction enzymes which specifically cleave sites created or retained due to the presence of methylated CpGs. None ofthe regions was methylated in normal colon, consistent with a uniform protection against de-novo methylation.
  • Regions 1 and 2 were frequently methylated in cancer cell lines, and behaved in a concordant manner. These 2 regions were methylated in most cancer cell types except gliomas, and most cell lines where methylation was found methylated both regions simultaneously. Region 3, which is less CG rich than any ofthe other regions, had either no methylation or very low levels of methylation in most cell lines. Regions 5, 6, and 7 behaved quite differently compared to 1-3. Methylation of these regions was less frequent than regions 1-2, as 22/35 cell lines had no detectable methylation there, despite often showing methylation of region 1-2. However, when methylation was present (in 13/35 cell lines), it affected all 3 regions simultaneously, although to varying extents.
  • regions 4 and 8 behaved differentially again, being partially methylated primarily in colon and breast cell lines. Therefore, with regards to hypermethylation in cancer, the CpG rich region upstream of CACNAIG appears to be composed of 2 CpG islands which behave independently. MINT31 corresponds to the upstream CpG island (island 1, regions 1 and 2), while the 5' region of CACNAIG is contained in the downstream CpG island (island 2, regions 5-7). Regions 3, 4 and 8 correspond to the edge of these CpG islands, and behave a little differentially than the hearts ofthe CpG islands, as previously described for the E-Cad gene (Graff, et al., J. Biol. Chem. 221: 22322-22329, 1997).
  • methylation patterns fell into 5 distinct categories: (1) No methylation in any region (normal tissue). (2) Slight methylation of island 1 (6 cell lines, see for example TSU-PRL in Fig. 2). (3) Heavy methylation of island 1 but no methylation of island 2 (16 cell lines, see for example Caco2 in Fig. 2). (4) Heavy methylation of island 1 and moderate to heavy methylation of island 2 (6 cell lines, see for example RKO and Raji in Fig. 2). (5) High methylation of island 1 and low to moderate methylation of island 2 (7 cell lines, see for example MB-231 in Fig. 2).
  • CACNAIG CACNAIG was expressed ubiquitously in a variety of tissues and cell lines. In normal tissues expression was relatively low but easily detectable, while most cell lines had relatively high expression of CACNAIG. However, some cell lines had negligible or totally absent levels of CACNAIG expression. The results of CACNAIG expression was co ⁇ elated with the detailed methylation analysis previously described.
  • CACNAIG CpG island methylator phenotype
  • CACNAIG affects the expression status of this gene in primary tumors, we performed RT-PCR using cDNA from a series of colorectal adenomas. Six out of 8 cases which showed no methylation of region 7 expressed CACNAIG. In sharp contrast, all 5 cases that showed methylation of region 7 had no detectable expression of this gene.
  • a human T-type calcium channel gene (CACNAIG) has been identified and cloned using the MINT31 sequence as a probe.
  • the human T- type calcium channel gene has been determined to be a target of aberrant methylation and silencing in human tumors.
  • the data show that MINT31 (a representative sequence of MINT 1-33) can be used as a probe to identify genes that play a role in disorders such as cell proliferative disorders.
  • the CACNAIG promoter is contained in a large CG rich area that is not coordinately methylated in cancer.
  • the CpG island around MINT31 is much more frequently methylated in cancers compared to that just upstream of CACNAIG. This may simply be caused by differential susceptibility to de-novo methylation between these two regions, with methylation of MINT31 serving as a trigger, and eventually spreading to CACNAIG, as described in other genes (Graff, et al., J Biol. Chem. 272: 22322-22329, 1997).
  • MINT31 methylation of MINT31 appears to be independent of methylation of CACNAIG suggesting that they are 2 distinct CpG islands regulated by different mechanisms. These data leave open the possibility that MINT31 is the promoter for an unidentified gene, which may perhaps be transcribed opposite to CACNAIG.
  • T-type calcium channels are involved not only in electrophysiological rhythm generation but also in the control of cytosolic calcium during cell proliferation and cell death (reviewed in Berridge, et al., Nature 395: 645- 648, 1998).
  • the results demonstrate that the expression of CACNAIG is not limited to brain and heart, suggesting that it may play a role in these other tissues. It has previously been shown that Ca 2+ influx via T-type channels is an important factor during the initial stages of cell death such as apoptosis (Berridge, et al., Nature 2 1: 645-648, 1998), ischemia (Fern, J. Neurosci.
  • RXMA and RMCA PCR adaptors were prepared by incubation ofthe oligonucleotides RXMA24 (5'-AGCACTCTCCAGCCTCTCACCGAC-3') (SEQ ID NO: 34) and RXMA12 (5'-CCGGGTCGGTGA-3') (SEQ ID NO:35), or RMCA24 (5'-CCACCGCCATCCGAGCCTTTCTGC-3') (SEQ ID NO:36) and RMCA12 (5'-CCGGGCAGAAAG-3') (SEQ ID NO:37) at 657C for two min. followed by cooling to room temperature. 0.5 ⁇ g of DNA was ligated to 0.5 nmol of RXMA or RMCA adaptor using T4 DNA ligase (NEB).
  • NEB T4 DNA ligase
  • PCR was performed using 3 ⁇ l of each ofthe ligation mix as a template in a 100 ⁇ l volume containing 100 pmol of RXA24 or RMC24 primer, 5 units of Taq DNA polymerase, (GIBCO-BRL.), 4 mM MgC12, 16 mM of NH4 (SO 4 )2, lOmg/ml of BSA, and 5% v/v DMSO.
  • the reaction mixture was incubated at 72 C at 5 min and at 95 C for 3 min. Samples were then subjected to 25 cycles of amplification consisting of 1 min at 95°/C, and 3 min either at 72 C or 777C in a thermal cycler (Hybaid, Inc.). The final extension time was 10 min.
  • MCA products from normal colon mucosa and corresponding cancer tissues were prepared as described above.
  • One ⁇ g of MCA products was resuspended in 4 ⁇ l of TE (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0), mixed with 2 ⁇ l of 20 X SSC, and 1 ⁇ l aliquot of this mix was blotted onto nylon membranes (Nunc) using a 96 well replication system (Nunc).
  • the membranes were baked at 807C, UN crosslinked for 2 min. and hybridized using 32 P labeled probes. Each sample was blotted in duplicate.
  • Each filter included mixtures of a positive control (Caco2) and a negative control (normal colon mucosa from an 18 year old individual).
  • the filters were exposed to a phosphor screen for 24 to 72 hours and developed using a phosphorimager (Molecular Dynamics). The intensity of each signal was calculated using the Image Quant software, and methylation levels were determined relative to the control samples.
  • RDA was performed essentially as previously reported (Lisitsyn et al, 1993) with the following modifications.
  • 500 ng and 100 ng of ligation mix was used, respectively.
  • a cD ⁇ A spun column was used instead of excising from the agarose gel.
  • Primers used for the first and second rounds of RDA are as follows :
  • PCR products were digested with Xmal.
  • the J adaptor was eliminated by column filtration.
  • the PCR products were then subcloned into Bluescript SK(-) (Stratagene).
  • Bluescript SK(-) (Stratagene).
  • a total of 396 clones were cultured overnight in LB medium with ampicillin and 3 ⁇ l ofthe culture was directly used as template for a PCR reaction.
  • Each clone was amplified with
  • T3 (5'-AATTAACCCTCACTAAAGGG-3') (SEQ ID NO:46) and T7 (5'-GTAATACGACTCACTATAGGGC-3 ') (SEQ ID NO:47) primers, blotted onto nylon membranes, and screened for cross hybridization with 32P labeled inserts.
  • the clones differentially hybridizing to tester and driver MCA products were further characterized by Southern blot analysis and DNA sequencing.
  • Plasmid DNA was prepared using the Wizard Plus Minipreps (Promega) according to the suppliers recommendation. Sequence analysis was carried out at the Johns Hopkins Core Sequencing Facility using automated DNA sequencers (Applied Biosystems). Sequence homologies were identified using the BLAST program ofthe National Center for Biotechnology Information (NCBI) available at http://www.ncbi.nlm.nih.gov/BLAST using the default parmaters ofthe web site. Putative promoter sequences were predicted using the computer programs NNPP and TSSG available through the Baylor college of Medicine launcher at http://dot.imgen.bcm.tmc.edu:9331.
  • NCBI National Center for Biotechnology Information
  • the PCR products were visualized by ethidium bromide staining.
  • the cDNA samples were also amplified using GAPDH gene, primers
  • chromosomal location of clones that did not co ⁇ espond to known genes was determined using a human-rodent somatic cell hybrid panel and a radiation hybrid panel (Research Genetics). PCR reactions were performed using 30 ng of each ofthe hybrid panel DNA as a template in a 40 ⁇ l volume containing 15 pmol of each primer, 0.5 units of Taq DNA polymerase, (GIBCO BRL), 2mM MgC12, BSA and 5% DMSO. First denaturation was carried out at 957C for 3 min. Samples were then subjected to 35 cycles of amplification consisting of 25 sec. at 947C, 1 min at 60 to 687C and 1.5 min. at 727C in a thermal cycler (Hybaid).
  • the final extension time was 10 min.
  • Ten ⁇ l ofthe PCR product were electrophoresed in a 2 % agarose and the genotype of each panel was determined. Linkage analysis was performed using the RH server of Stanford University as described (Stewart et al. , 1997).

Abstract

A method is provided for identifying a methylated CpG containing nucleic acid including contacting a nucleic acid with a methylation sensitive restriction endonuclease that cleaves unmethylated CpG sites, contacting the sample with an isoschizomer of the methylation sensitive restriction endonuclease, wherein the isoschizomer cleaves both methylated and unmethylated CpG sites. The method also includes adding an oligonucleotide under conditions and for a time to allow ligation of the oligonucleotide to nucleic acid cleaved by the restriction endonuclease, and amplifying the nucleic acid. A method is also provided for detecting an age associated disorder including contacting a nucleic acid with a methylation sensitive restriction endonuclease that cleaves unmethylated CpG sites, contacting the sample with an isoschizomer of the methylation sensitive restriction endonuclease, wherein the isoschizomer cleaves both methylated and unmethylated CpG sites. The method also includes adding an oligonucleotide under conditions and for a time to allow ligation of the oligonucleotide to nucleic acid cleaved by the restriction endonuclease, and amplifying the nucleic acid. The amplified digested nucleic acid is adhered to a membrane, and hybridized with a probe of interest. A kit useful for detection of a CpG containing nucleic acid is also provided.

Description

METHYLATED CpG ISLAND AMPLIFICATION (MCA)
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made with Government support under Grant No. CA43318 and CA54396, awarded by the National Cancer Institute and Grant No. CA43318, a Colon Cancer Spore Grant. The government may have certain rights in the invention.
FIELD OF THE INVENTION
The present invention relates generally to regulation of gene expression and more specifically to a method of determining the DNA methylation status of CpG sites in a given locus.
BACKGROUND OF THE INVENTION
DNA methylases transfer methyl groups from the universal methyl donor S-adenosyl methionine to specific sites on the DNA. Several biological functions have been attributed to the methylated bases in DNA. The most established biological function for methylated DNA is the protection of DNA from digestion by cognate restriction enzymes. The restriction modification phenomenon has, so far, been observed only in bacteria. Mammalian cells, however, possess a different methylase that exclusively methylates cytosine residues that are 5' neighbors of guanine (CpG). This modification of cytosine residues has important regulatory effects on gene expression, especially when involving CpG rich areas, known as CpG islands, located in the promoter regions of many genes.
Methylation has been shown by several lines of evidence to play a role in gene activity, cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes (Razin, A., H., and Riggs, R.D. eds. in DNA Methylation Biochemistry and Biological Significance. Springer- Verlag, New York, 1984). Methylation has been shown by several lines of evidence to play a role in gene activity, cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes (Razin, A. H. and Riggs, R.D. eds. in DNA Methylation Biochemistry and Biological Significance. Springer-Verlag. New York. 1984V In eukaryotic cells, methylation of cytosine residues that are immediately 5' to a guanosine, occurs predominantly in CG poor regions (Bird, A., Nature, 121:209, 1986). In contrast, CpG islands remain unmethylated in normal cells, except during X-chromosome inactivation (Migeon, et al, supra) and parental specific imprinting (Li, et al, Nature, :362, 1993) where methylation of 5' regulatory regions can lead to transcriptional repression. De novo methylation of the Rb gene has been demonstrated in a small fraction of retinoblastomas (Sakai, et al, Am. J. Hum. Genet., 48:880, 1991), and recently, a more detailed analysis of the VHL gene showed aberrant methylation in a subset of sporadic renal cell carcinomas (Herman, et al, Proc. Natl. Acad. Sci., U.S.A., 91 :9700. 1994). Expression of a tumor suppressor gene can also be abolished by de novo DNA methylation of a normally unmethylated CpG island (Issa, et al, Nature Genet., 2:536, 1994; Herman, et al, supra; Merlo, et al, Nature Med, 1:686, 1995; Herman, et al, Cancer Res., 56:722, 1996; Graff, et al, Cancer Res., 5 5:5195, 1995; Herman, et al, Cancer Res., 5_£:4525. 1995).
Human cancer cells typically contain somatically altered nucleic acid, characterized by mutation, amplification, or deletion of critical genes. In addition, the nucleic acid from human cancer cells often displays somatic changes in DNA methylation (E.R. Fearon, et al, Cell, 61:759, 1990;
P.A. Jones, et al, Cancer Res., 46:461, 1986; R. Holliday, Science, 23 _:163, 1987; A. De Bustros, et al, Proc. Natl. Acad. Sci., USA, :5693, 1988); P. A Jones, et al, Adv. Cancer Res., 54:1, 1990; S.B. Baylin, et al, Cancer Ce/&_2:383, 1991; M. Makos, et al, Proc. Natl. Acad. Sci., USA, £9:1929, 1992; N. Ohtani-Fujita, et al, Oncogene, £:1063, 1993). However, the precise role of abnormal DNA methylation in human tumorigenesis has not been established. Aberrant methylation of normally unmethylated CpG islands has been described as a frequent event in immortalized and transformed cells, and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers. In the development of colorectal cancers (CRC), a series of tumor suppressor genes (TSG) such as APC, p53, DCC and DPC4 are inactivated by mutations and chromosomal deletions (reviewed in Kinzler and Vogelstein 1996). Some of these alterations result from a chromosomal instability phenotype described in a subset of CRC (Lengauer et al, 1997a). Recently, an additional pathway has been shown to be involved in a familial form of CRC, hereditary non-polyposis colorectal cancer. The cancers from these patients show a characteristic mutator phenotype which causes microsatellite instability (MI), and mutations at other gene loci such as TGF-β-RII (Markowitz et al. , 1995) and BAX (Rampino et al. , 1997). This phenotype usually results from mutations in the mismatch repair (MMR) genes hMSH2 and hMLHl (reviewed by Peltomaki, and de la Chapelle, 1997). A subset of sporadic CRC also show MI, but mutations in MMR genes appear to be less frequent in these tumors (Liu et al, 1995; Moslein et al, 1996).
Another molecular defect described in CRC is CpG island (CGI) methylation. CGIs are short sequences rich in the CpG dinucleotide and can be found in the 5' region of about half of all human genes (Bird, 1986). Methylation of cytosine within 5' CGIs is associated with loss of gene expression and has been seen in physiological conditions such as X chromosome inactivation and genomic imprinting (reviewed in Latham, 1996). Aberrant methylation of CGIs has been detected in genetic diseases such as the fragile-X syndrome (Hansen et al, 1992), in aging cells (Issa et al, 1994) and in neoplasia. About half of the tumor suppressor genes which have been shown to be mutated in the germline of patients with familial cancer syndromes have also been shown to be aberrantly methylated in some proportion of sporadic cancers, including Rb, VHL, pi 6, hMLHI, and BRCAl (reviewed in Baylin et al, 1998; Jones 1997). TSG methylation in cancer is usually associated with (1) lack of gene transcription and (2) absence of coding region mutation. Thus it has been proposed that CGI methylation serves as an alternative mechanism of gene inactivation in cancer.
The causes and global patterns of CGI methylation in human cancers remain poorly defined. Aging could play a factor in this process because methylation of several CGIs could be detected in an age-related manner in normal colon mucosa as well as in CRC (Issa et al, 1994). In addition, aberrant methylation of CGIs has been associated with the MI phenotype in CRC (Ahuja et al, 1997) as well as specific carcinogen exposures (Issa et al, 1996). However, an understanding of aberrant methylation in CRC has been somewhat limited by the small number of CGIs analyzed to date. In fact, previous studies have suggested that large numbers of CGIs are methylated in immortalized cell lines (Antequera et al, 1990), and it is not well understood whether this global aberrant methylation is caused by the cell culture conditions or whether they are an integral part of the pathogenesis of cancer.
Most of the methods developed to date for detection of methylated cytosine depend upon cleavage of the phosphodiester bond alongside cytosine residues, using either methylation-sensitive restriction enzymes or reactive chemicals such as hydrazine which differentiate between cytosine and its 5- methyl derivative. Genomic sequencing protocols which identify a 5-MeC residue in genomic DNA as a site that is not cleaved by any of the Maxam Gilbert sequencing reactions have also been used, but still suffer disadvantages such as the requirement for large amount of genomic DNA and the difficulty in detecting a gap in a sequencing ladder which may contain bands of varying intensity.
Mapping of methylated regions in DNA has relied primarily on Southern hybridization approaches, based on the inability of methylation- sensitive restriction enzymes to cleave sequences which contain one or more methylated CpG sites. This method provides an assessment of the overall methylation status of CpG islands, including some quantitative analysis, but is relatively insensitive and requires large amounts of high molecular weight DNA.
Another method utilizes bisulfite treatment of DNA to convert all unmethylated cytosines to uracil. The altered DNA is amplified and sequenced to show the methylation status of all CpG sites. However, this method is technically difficult, labor intensive and without cloning amplified products, it is less sensitive than Southern analysis, requiring approximately 10% of the alleles to be methylated for detection.
Identification of the earliest genetic changes in tumorigenesis is a major focus in molecular cancer research. Diagnostic approaches based on identification of these changes are likely to allow implementation of early detection strategies and novel therapeutic approaches targeting these early changes might lead to more effective cancer treatment. SUMMARY OF THE INVENTION
The invention provides a method for detecting a methylated CpG- containing nucleic acid. This method can be used to identify sequences which are differentially methylated during a disease process such as a cell proliferative disorder.
In one embodiment, a method is provided for identifying a methylated CpG-containing nucleic acid. The method includes contacting a nucleic acid sample suspected of containing a CpG-containing nucleic acid, with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites, under conditions and for a time to allow cleavage of unmethylated nucleic acid; and contacting the sample with an isoschizomer of the methylation sensitive restriction endonuclease, wherein the isoschizomer of the methylation sensitive restriction endonuclease cleaves both methylated and unmethylated CpG sites. Oligonucleotides are added to the nucleic acid sample under conditions and for a time to allow ligation of the oligonucleotides to nucleic acid cleaved by the restriction endonuclease and the digested nucleic acid is amplified for further analysis.
In another embodiment, a method is provided for detecting an age- associated disorder associated with methylation of CpG islands in a nucleic acid sequence of interest in a subject having or at risk of having said disorder. The method includes contacting a nucleic acid sample suspected of comprising a CpG-containing nucleic acid with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites under conditions and for a time to allow cleavage of unmethylated nucleic acid, and contacting the sample with an isoschizomer of the methylation sensitive restriction endonuclease, wherein the isoschizomer of the methylation sensitive restriction endonuclease cleaves both methylated and unmethylated CpG sites. Oligonucleotides are added to the nucleic acid sample under conditions and for a time to allow ligation of the oligonucleotides to nucleic acid cleaved by the restriction endonuclease, and the digested nucleic acid is amplified. The amplified, digested nucleic acid is contacted with a membrane and the membrane is hybridized with a probe of interest.
In yet another embodiment, a method is provided for evaluating the response of a cell to an agent. The method includes contacting a nucleic acid sample suspected of containing a CpG-containing nucleic acid with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites, under conditions and for a time to allow cleavage of unmethylated nucleic acid, and contacting the sample with an isoschizomer of the methylation sensitive restriction endonuclease, wherein the isoschizomer of the methylation sensitive restriction endonuclease cleaves both methylated and unmethylated CpG sites. Oligonucleotides are added to the nucleic acid sample under conditions and for a time to allow ligation of the oligonucleotides to nucleic acid cleaved by the restriction endonuclease, and the digested nucleic acid is amplified. The amplified, digested nucleic acid is adhered to a membrane and the membrane is hybridized with a probe of interest.
In a further embodiment, a kit for the detection of a methylated CpG- containing nucleic acid is provided. In one embodiment the kit includes a carrier means containing one or more containers including a container containing an oligonucleotide for ligation of the oligonucleotides to nucleic acid, a second container containing a methylation sensitive restriction endonuclease and a third container containing an isoschizomer of the methylation sensitive endonuclease. In another embodiment the kit includes a carrier means containing one or more containers containing a membrane, wherein the membrane has a member of the group consisting SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 (MINT1, MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINT14, MINT15, MINT17, MINT19, MINT20, MINT22, MINT23,
MINT24, MINT27, MINT30, MINT31, MINT32, and MINT33 immobilized on the membrane.
In a further embodiment, an isolated nucleic acid including a member selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 (MINT1, MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINT14, MINT15, MINT17, MINT19, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32, and MINT33) is provided. An isolated methylated nucleic acid sequence having a sequence as set forth in a member of the group consisting of SEQ ID NOs:l-33 (MINT1-33) is also provided. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of MCA. A hypothetical fragment of genomic DNA is represented by a solid line, with 7 Smal sites depicted by tick marks. Methylated Smal sites are indicated by an m. Fragments B and D are CpG islands. B is methylated in both normal (right) and cancer (left), while D is differentially methylated in cancer. For MCA, unmethylated Smal sites are eliminated by digestion with Smal (which is methylation-sensitive and does not cleave when its recognition sequence CCCGGG contains a methylated CpG), which leaves the fragment blunt ended. Methylated Smal sites are then digested with the non-methylation sensitive Smal isoschizomer Xmal, which digests methylated CCCGGG sites, leaving a CCGG overhang (sticky ends). Adaptors are ligated to these sticky ends, and PCR is performed to amplify the methylated sequences. The MCA amplicons can be used directly in a dot blot analysis to study the methylation status of any gene for which a probe is available (left). Alternatively, MCA products can be used to clone differentially methylated sequence by RDA (right).
FIG. 2 shows an the nucleotide sequence of a differentially Methylated Clone, MINT2 obtained by MCA Followed by RDA. The restriction endonuclease sites for Smal are underlined. Primer sequences used for bisulfite-PCR are also underlined. The restriction endonuclease site for BstUI used to detect methylation after bisulfite PCR is shown by a gray box.
FIG. 3 show a map of the versican gene first exon (filled box) and flanking regions. The position of MINTl 1 is shown by a solid line (on top). CpG sites are indicated below. Location of the primers used for bisulfite-PCR are shown by arrows. FIG. 4 is a pictorial representation of global hypermethylation in CRC. Each column represents a separate gene locus. Each row is a primary colorectal cancer (samples above the bold solid line) or polyp ( below the bold solid line). Black squares: methylation > 10%. Gray squares: 1-10% methylation. White squares: < 1% methylation. A: GH+MI+, B: GH+MI-, C: GH-MI+, D: GH-MI-, E: GH+, F: GH-. A-D are cancers. E and F are adenomas. MI denotes the presence of microsatellite instability. ND, not done.
FIG. 5 shows a model integrating CGI methylation in colorectal carcinogenesis.
FIGS. 6A-H are the nucleic acid sequences of MINT1-33 (SEQ ID NO: 1-33).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides a method for identifying a methylated CpG-containing nucleic acid called methylated CpG island amplification (MCA). MCA can be used to study methylation in normal and neoplastic cells, and allows rapid screening of nucleic acid samples for the presence of hypermethylation of specific genes. MCA can also be used to clone genes and nucleic acid sequences differentially methylated in normal and abnormal tissues and cells.
It should be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the restriction enzyme" includes reference to one or more restriction enzymes and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the prefeπed methods, devices and materials are now described.
All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the methodologies which are described in the publications which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.
Any nucleic acid sample, in purified or nonpurified form, can be utilized as the starting nucleic acid or acids, provided it contains, or is suspected of containing, a nucleic acid sequence containing the target locus (e.g., CpG-containing nucleic acid). In general the CpG-containing nucleic acid will be DNA. However, the process may employ, for example, samples that contain DNA, or DNA and RNA, including messenger RNA, wherein DNA or RNA may be single stranded or double stranded, or a DNA-RNA hybrid may be included in the sample. A mixture of nucleic acids may also be employed. The specific nucleic acid sequence to be detected may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be studied be present initially in a pure form; the nucleic acid may be a minor fraction of a complex mixture, such as contained in whole human DNA. The nucleic acid may be contained in a biological sample. Such samples include but are not limited to a serum, urine, saliva, cerebrospinal fluid, pleural fluid, ascites fluid, sputum, stool, or biopsy sample. The nucleic acid-containing sample used for detection of methylated CpG may be from any source including, but not limited to, brain, colon, urogenital, hematopoietic, thymus, testis, ovarian, uterine, prostate, breast, colon, lung and renal tissue and may be extracted by a variety of techniques such as that described by Maniatis, et al. ^Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY, pp 280, 281, 1982).
The nucleic acid of interest can be any nucleic acid where it is desirable to detect the presence of a CpG island. In one embodiment, the CpG island comprises a CpG island located in a gene. A "CpG island" is a CpG rich region of a nucleic acid sequence. The nucleic acid sequence may be, for example, a pl6, a Rb, a VHL, a hMLHI, or a BRCAl gene. Alternatively the nucleic acid of interest can be, for example, a MINTl -33 nucleic acid sequence. However, any gene or nucleic acid sequence of interest containing a CpG sequence can be detected using the method of the invention.
The presence of methylated CpG in the nucleic acid-containing specimen may be indicative of a disorder. In one embodiment, the disorder is a cell proliferative disorder. A "cell proliferative disorder" is any disorder in which the proliferative capabilities of the affected cells is different from the normal proliferative capabilities of unaffected cells. An example of a cell proliferative disorder is neoplasia. Malignant cells (i.e., cancer) develop as a result of a multistep process. Specific, non-limiting examples of disorders associated with increased methylation of CpG-islands are colon cancer, lung cancer, renal cancer, leukemia, breast cancer, prostate cancer, uterine cancer, astrocytoma, glioblastoma, and neuroblastoma.
In another embodiment, the disorder is an age-associated disorder. The term "age-associated disorder" is used to describe a disorder observed with the biological progression of events occurring over time in a subject. Preferably, the subject is a human. Non-limiting examples of age-associated disorders include, but are not limited to, atherosclerosis, diabetes melitis, and dementia. An age-associated disorder may also be a cell proliferative disorder. Examples of age-associated disorders which are cell proliferative disorders include colon cancer, lung cancer, breast cancer, prostate cancer, and melanoma, amongst others. An age-associated disorder is further intended to mean the biological progression of events that occur during a disease process that affects the body, which mimic or substantially mimic all or part of the aging events which occur in a normal subject, but which occur in the diseased state over a shorter period of time.
In one embodiment, the age-associated disorder is a "memory disorders or learning disorders" which are characterized by a statistically significant decrease in memory or learning assessed over time by the Randt Memory Test (Randt et al, Clin. Neuropsychol. , 2:184, 1980), Wechsler Memory Scale (J Psych., 12:87-95, 1945), Forward Digit Span test (Craik, Age Differences in Human Memory, in: Handbook of the Psychology of Aging. Birren, J., and Schaie, K., Eds., New York, Van Nostrand, 1977), Mini-Mental State Exam (Folstein et al, J. of Psych. Res. 12:189-192, 1975), or California Verbal Learning Test (CVLT) wherein such non-neurodegenerative pathological factors as aging, anxiety, fatigue, anger, depression, confusion, or vigor are controlled for. (See, U.S. Patent No. 5,063,206 for example).
If the sample is impure (e.g., plasma, serum, stool, ejaculate, sputum, saliva, cerebrospinal fluid or blood or a sample embedded in paraffin), it may be treated before amplification with a reagent effective for opening the cells, fluids, tissues, or animal cell membranes of the sample, and for exposing the nucleic acid(s). Methods for purifying or partially purifying nucleic acid from a sample are well known in the art (e.g. , Sambrook et al. , Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Press, 1989, herein incorporated by reference).
In one embodiment, a method is provided for identifying a methylated
CpG-containing nucleic acid, including contacting a nucleic acid sample suspected of comprising a CpG-containing nucleic acid with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites under conditions and for a time to allow cleavage of unmethylated nucleic acid. The sample is further contacted with an isoschizomer of the methylation sensitive restriction endonuclease, that cleaves both methylated and unmethylated CpG-sites, under conditions and for a time to allow cleavage of methylated nucleic acid. Oligonucleotides are added to the nucleic acid sample under conditions and for a time to allow ligation of the oligonucleotides to nucleic acid cleaved by the restriction endonuclease, and the digested nucleic acid is amplified. Following identification, the methylated CpG-containing nucleic acid can be cloned, using method well known to one of skill in the art (see Sambrook et al. , Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Press, 1989).
A "methylation sensitive restriction endonuclease" is a restriction endonuclease that includes CG as part of its recognition site and has altered activity when the C is methylated as compared to when the C is not methylated. Preferably, the methylation sensitive restriction endonuclease has inhibited activity when the C is methylated (e.g., Smal). Specific non-limiting examples of a methylation sensitive restriction endonucleases include Smal, BssHll, or Hpall. Such enzymes can be used alone or in combination. Other methylation sensitive restriction endonucleases will be known to those of skill in the art and include, but are not limited to SαcII, Eagl, and BstUl, for example. An "isoschizomer" of a methylation sensitive restriction endonuclease is a restriction endonuclease which recognizes the same recognition site as a methylation sensitive restriction endonuclease but which cleaves both methylated and unmethylated CGs. One of skill in the art can readily determine appropriate conditions for a restriction endonuclease to cleave a nucleic acid (see Sambrook et al, Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Press, 1989). Without being bound by theory, actively transcribed genes generally contain fewer methylated CGs than in other genes.
In the method of the invention, a nucleic acid of interest is cleaved with a methylation sensitive endonuclease. In one embodiment, cleavage with the methylation sensitive endonuclease creates a sufficient overhang on the nucleic acid of interest. Following cleavage with the isoschizomer, the cleavage product can still have a sufficient overhang. An "overhang" refers to nucleic acid having two strands wherein the strands end in such a manner that a few bases of one strand are not base paired to the other strand. A "sufficient overhang" refers to an overhang of sufficient length to allow specific hybridization of an oligonucleotide of interest. In one embodiment, a sufficient overhang is at least two bases in length. In another embodiment, the sufficient overhang is four or more bases in length. An overhang of a specific sequence on the nucleic acid of interest may be desired in order for an oligonucleotide of interest to hybridize. In this case, the isoschizomer can be used to create the overhang having the desired sequence on the nucleic acid of interest.
In another embodiment, the cleavage with a methylation sensitive endonuclease results in a reaction product of the nucleic acid of interest that has a blunt end or an insufficient overhang. In this embodiment, an isoschizomer of the methylation sensitive restriction endonuclease can create a sufficient overhang on the nucleic acid of interest. "Blunt ends" refers to a flush ending of two stands, the sense stand and the antisense strand, of a nucleic acid.
Once a sufficient overhang is created on the nucleic acid of interest, an oligonucleotide is ligated to the nucleic acid cleaved of interest which has been cleaved by the methylation specific restriction endonuclease. "Ligation" is the attachment of two nucleic acid sequences by base pairing of substantially complementary sequences or by the formation of a covalent bonds between two nucleic acid sequences. An "oligonucleotide" is a nucleic acid sequence of 2 to 40 bases in length. Preferably the oligonucleotide is from 15 to 35 bases in length. In one embodiment, the oligonucleotide is ligated to the overhang on the nucleic acid sequence of interest by base pairing. In one embodiment, two oligonucleotides are utilized to form an adaptor. An "adaptor" is a double-stranded nucleic acid sequence with one end that has a sufficient single-stranded overhang at one or both ends such that the adaptor can be ligated by base-pairing to a sufficient overhang on a nucleic acid of interest that has been cleaved by a methylation sensitive restriction enzyme or an isoschizomer of a methylation sensitive restriction enzyme. In one embodiment, two oligonucleotides can be used to form an adaptor; these oligonucleotides are substantially complementary over their entire sequence except for the region(s) at the 5' and/or 3' ends that will form a single stranded overhang. The single stranded overhang is complementary to an overhang on the nucleic acid cleaved by a methylation sensitive restriction enzyme or an isoschizomer of a methylation sensitive restriction enzyme, such that the overhang on the nucleic acid of interest will base pair with the 3 ' or 5' single stranded end of the adaptor under appropriate conditions. The conditions will vary depending on the sequence composition (GC vs AT), the length, and the type of nucleic acid (see Sambrook et al, Molecular Cloning: a Laboratory Manual. 2nd Ed.; Cold Spring Harbor Laboratory Press, Plainview, NY, 1998).
Following the ligation of the oligonucleotide, the nucleic acid of interest is amplified using a primer complementary to the oligonucleotide. Specifically, the term "primer" as used herein refers to a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and most preferably more than 8, which sequence is capable of initiating synthesis of a primer extension product, which is substantially complementary to a nucleic acid such as an adaptor or a ligated oligonucleotide. Environmental conditions conducive to synthesis include the presence of nucleoside triphosphates and an agent for polymerization, such as DNA polymerase, and a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency in amplification, but may be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. In one embodiment, the primer is an oligodeoxyribo-nucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent for polymerization. The exact length of primer will depend on many factors, including temperature, buffer, and nucleotide composition. The oligonucleotide primer typically contains 12-20 or more nucleotides, although it may contain fewer nucleotides.
Primers of the invention are designed to be "substantially" complementary to each strand of the oligonucleotide to be amplified and include the appropriate G or C nucleotides as discussed above. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions which allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with a 5' and 3' oligonucleotide to hybridize therewith and permit amplification of CpG containing nucleic acid sequence.
Primers of the invention are employed in the amplification process which is an enzymatic chain reaction that produces exponential quantities of target locus relative to the number of reaction steps involved (e.g., polymerase chain reaction or PCR). Typically, one primer is complementary to the negative (-) strand of the locus and the other is complementary to the positive (+) strand. Annealing the primers to denatured nucleic acid followed by extension with an enzyme, such as the large fragment of DNA Polymerase I (Klenow) and nucleotides, results in newly synthesized + and - strands containing the target locus sequence. Because these newly synthesized sequences are also templates, repeated cycles of denaturing, primer annealing, and extension results in exponential production of the region (i.e., the target locus sequence) defined by the primer. The product of the chain reaction is a discrete nucleic acid duplex with termini corresponding to the ends of the specific primers employed.
The oligonucleotide primers of the invention may be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof. In one such automated embodiment, diethylphosphoramidites are used as starting materials and may be synthesized as described by Beaucage, et al. (Tetrahedron Letters, 22: 1859-1862, 1981). One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066.
Where the CpG-containing nucleic acid sequence of interest contains two strands, it is necessary to separate the strands of the nucleic acid before it can be used as a template for the amplification process. Strand separation can be effected either as a separate step or simultaneously with the synthesis of the primer extension products. This strand separation can be accomplished using various suitable denaturing conditions, including physical, chemical, or enzymatic means, the word "denaturing" includes all such means. One physical method of separating nucleic acid strands involves heating the nucleic acid until it is denatured. Typical heat denaturation may involve temperatures ranging from about 80° to 105°C for times ranging from about 1 to 10 minutes. Strand separation may also be induced by an enzyme from the class of enzymes known as helicases or by the enzyme RecA, which has helicase activity, and in the presence of riboATP, is known to denature DNA. The reaction conditions suitable for strand separation of nucleic acids with helicases are described by Kuhn Hoffmann-Berling (CSH-Quantitative Biology, 43:63, 1978) and techniques for using RecA are reviewed in C. Radding (Ann. Rev. Genetics, 16:405-437, 1982).
When complementary strands of nucleic acid or acids are separated, regardless of whether the nucleic acid was originally double or single stranded, the separated strands are ready to be used as a template for the synthesis of additional nucleic acid strands. This synthesis is performed under conditions allowing hybridization of primers to templates to occur. Generally synthesis occurs in a buffered aqueous solution, generally at a pH of about 7- 9. Preferably, a molar excess (for genomic nucleic acid, usually about 108:1 primeπtemplate) of the two oligonucleotide primers is added to the buffer containing the separated template strands. It is understood, however, that the amount of complementary strand may not be known if the process of the invention is used for diagnostic applications, so that the amount of primer relative to the amount of complementary strand cannot be determined with certainty. As a practical matter, however, the amount of primer added will generally be in molar excess over the amount of complementary strand (template) when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands. A large molar excess is preferred to improve the efficiency of the process.
The deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to about 90°- 100°C from about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period, the solution is allowed to cool to approximately room temperature, which is preferable for the primer hybridization. To the cooled mixture is added an appropriate agent for effecting the primer extension reaction (called herein "agent for polymerization"), and the reaction is allowed to occur under conditions known in the art. The agent for polymerization may also be added together with the other reagents if it is heat stable. This synthesis (or amplification) reaction may occur at room temperature up to a temperature above which the agent for polymerization no longer functions. Thus, for example, if DNA polymerase is used as the agent, the temperature is generally no greater than about 40°C. Most conveniently the reaction occurs at room temperature.
The agent for polymerization may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, polymerase muteins, reverse transcriptase, and other enzymes, including heat-stable enzymes (i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation). Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each locus nucleic acid strand. Generally, the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths. There may be agents for polymerization, however, which initiate synthesis at the 5' end and proceed in the other direction, using the same process as described above. Preferably, the method of amplifying is by PCR, as described herein and as is commonly used by those of ordinary skill in the art. However, alternative methods of amplification have been described and can also be employed.
Once amplified, the nucleic acid can be attached to a solid support, such as a membrane, and can be hybridized with any probe of interest, to detect any nucleic acid sequence. Several membranes are known to one of skill in the art for the adhesion of nucleic acid sequences. Specific non- limiting examples of these membranes include nitrocellulose (Nitropure) or other membranes used in for detection of gene expression such as polyvinylchloride, diazotized paper and other commercially available membranes such as Genescreen™, Zetaprobe™ (Biorad), and Nytran™. Methods for attaching nucleic acids to these membranes are well known to one of skill in the art. Alternatively, screening can be done in a liquid phase.
In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
An example of progressively higher stringency conditions is as follows: 2 x SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68°C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each ofthe conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all ofthe steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically. In general, conditions of high stringency are used for the hybridization ofthe probe of interest.
The probe of interest can be detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme. Those of ordinary skill in the art will know of other suitable labels for binding to the probe, or will be able to ascertain such, using routine experimentation.
In one embodiment, representational difference analysis (RDA, see Lisitsyn et al, Science 259:946-951. 1993, herein incorporated by reference) can be performed on CpG-containing nucleic acid following MCA. MCA utilizes kinetic and subtractive enrichment to purify restriction endonuclease fragments present in one population of nucleic acid fragments but not in another. Thus, RDA enables the identification of small differences between the sequences of two nucleic acid populations. RDA uses nucleic acid from one population as a "tester" and nucleic acid from a second population as a "driver," in order to clone probes for single copy sequences present in (or absent from) one ofthe two populations. In one embodiment, nucleic acid from a "normal" individual or sample, not having a disorder such as a cell- proliferative disorder is used as a "driver," and nucleic acid from an "affected" individual or sample, having the disorder such as a cell proliferative disorder is used as a "tester." In one embodiment, the nucleic acid used as a "tester" is isolated from an individual having a cell proliferative disorder such as colon cancer, lung cancer, renal cancer, leukemia, breast cancer, prostate cancer, uterine cancer, astrocytoma, glioblastoma, and neuroblastoma. The nucleic acid used as a "driver" is thus normal colon, normal lung, normal kidney, normal blood cells, normal breast, normal prostate, normal uterus, normal astrocytes, normal glial and normal neurons, respectively. In an additional embodiment, the nucleic acid used as a "driver" is isolated from an individual having a cell proliferative disorder such as colon cancer, lung cancer, renal cancer, leukemia, breast cancer, prostate cancer, uterine cancer, astrocytoma, glioblastoma, and neuroblastoma. The nucleic acid used as a "tester" is thus normal colon, normal lung, normal kidney, normal blood cells, normal breast, normal prostate, normal uterus, normal astrocytes, normal glial and normal neurons, respectively. One of skill in the art will readily be able to identify the "tester" nucleic acid useful with to identify methylated nucleic acid sequences in given "driver" population.
SCREENING AGENTS FOR AN EFFECT ON METHYLATION
The invention provides a method for identifying an agent which can affect methylation. An agent can affect methylation by either increasing or decreasing methylation. The method includes incubating an agent and a sample containing a CpG-containing polynucleotide under conditions sufficient to allow the components to interact, and measuring the effect ofthe compound on the methylation ofthe CpG-containing nucleic acid. In one embodiment, the sample is a cell expressing a polynucleotide of interest. In another embodiment, the sample is substantially purified nucleic acid.
"Substantially purified" nucleic acid is nucleic acid which has been separated from the cellular components which naturally accompany it, or from contaminating elements such as proteins, lipids, or chemical resins. Substantially pure nucleic acid can be extracted from any cell type, or can be chemically synthesized. Purity can be measured by any appropriate method, such as measuring the absorbance of light (e.g., A26o A280 ratio).
The nucleic acid can be identified by the methylated CpG island amplification, as described above. The methylation ofthe polynucleotide in the sample can then be compared to the methylation of a control sample not incubated with the agent. The effect ofthe agent on methylation of a polynucleotide can be measured by assessing the methylation ofthe polynucleotide by the methods ofthe invention. Alternatively, the effect of the agent on methylation of a polynucleotide can be measured by assessing the expression ofthe polynucleotide of interest. Means of measuring expression are well known to one of skill in the art (e.g., Northern blotting or RNA dot blotting, amongst others). The agents which affect methylation can include peptides, peptidomimetics, polypeptides, pharmaceuticals, and chemical compounds and biological agents. Psychotropic, antiviral, and chemotherapeutic compounds can also be tested using the method ofthe invention.
"Incubating" includes conditions which allow contact between the test agent and the cell of interest. "Contacting" includes in solution and solid phase. The test agent may also be a combinatorial library for screening a plurality of compounds. Agents identified in the method ofthe invention can be further cloned, sequenced, and the like, either in solution of after binding to a solid support, by any method usually applied to the isolation of a specific DNA sequence Molecular techniques for DNA analysis (Landegren et al. , Science 242:229-237. 1988) and cloning have been reviewed (Sambrook et al, Molecular Cloning: a Laboratory Manual. 2nd Ed.; Cold Spring Harbor Laboratory Press, Plainview, NY, 1998.
The sample can be any sample of interest. The sample may be a cell sample or a membrane sample prepared from a cell sample. Suitable cells include any host cells containing a nucleic acid including a CpG island. The cells can be primary cells or cells of a cell line.
In one embodiment, the agent is incubated with the sample of interest suspected of including a CpG-containing nucleic acid and methylation is evaluated by MCA. Thus, nucleic acid from the sample suspected of including a CpG-containing nucleic acid is contacted with a methylation sensitive restriction endonuclease which cleaves only unmethylated CpG sites under conditions and for a time to allow cleavage of unmethylated nucleic acid. An isoschizomer ofthe methylation sensitive restriction endonuclease is also utilized. An oligonucleotide is added to the nucleic acid sample under conditions and for a time to allow ligation ofthe oligonucleotide to nucleic acid cleaved by said restriction endonuclease, and the digested nucleic acid is amplified. The digested nucleic acid is adhered to a membrane, and the membrane is hybridized with a probe of interest. In one embodiment, representation difference analysis can also be performed.
KITS The materials for use in the assay ofthe invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means containing one or more container means such as vials, tubes, and the like, each ofthe container means comprising one ofthe separate elements to be used in the method. One ofthe container means can comprise a container containing an oligonucleotide for ligation to nucleic acid cleaved by a methylation sensitive restriction endonuclease. One or more container means can also be included comprising a primer complementary to the oligonucleotide. In addition, one or more container means can also be included which comprise a methylation sensitive restriction endonuclease. One or more container means can also be included containing an isoschizomer of said methylation sensitive restriction enzyme.
In another embodiment, the kit may comprise a carrier means containing one or more container means comprising a solid support, wherein the solid support has a nucleic acid sequence selected from the group consisting of MINT 1-33 immobilized on the solid support. In one embodiment, the solid support is a membrane. Several membranes are known to one of skill in the art for the adhesion of nucleic acid sequences. Specific non-limiting examples of these membranes include nitrocellulose (Nitropure) or other membranes used in for detection of gene expression such as polyvinylchloride, diazotized paper and other commercially available membranes such as Genescreen™, Zetaprobe™ (Biorad), and Nytran™. The MINTl -33 sequences immobilized on the solid support can then be hybridized to nucleic acid sequences produced by performing the MCA procedure on the nucleic acids of a sample of interest in order to determine if the nucleic acid sequences contained in the sample are methylated.
POLYNUCLEOTIDES AND POLYPEPTIDES
In another embodiment, the invention provides isolated MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polynucleotides ( SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively). These polynucleotides include DNA, cD A and RNA sequences which encode MINTl , MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINTl 5, MINTl 7, MINT19, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polypeptides. It is understood that naturally occurring, synthetic, and intentionally manipulated polynucleotides are included. For example, MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINT15, MINT17, MINT 19, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 nucleic acids may be subjected to site-directed mutagenesis. The nucleic acid sequence for MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 also includes antisense sequences, and sequences encoding dominant negative forms of MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MONTI 0, MINT14, MINT 15, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31 , MINT32 and MINT33.
The invention provides methylated and unmethylated forms of MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polynucleotides ( SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,
SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively). Methylated nucleic acid sequences are also provided which include MINT3, MINT5, MINT 7, MINTl 1, MINT12, MINTl 3, MINT16, MINT18, MINT21, MINT25, MINT26, MINT28, and MINT29 (SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29, respectively). It is understood that naturally occurring, synthetic, and intentionally manipulated polynucleotides are included.
The polynucleotides ofthe invention includes "degenerate variants" sequences that are degenerate as a result ofthe genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of a polypeptide encoded by the nucleotide sequence of SEQ ID NOs: 1-33 is functionally unchanged.
Specifically disclosed herein are methylated and unmethylated isolated polynucleotide sequences of MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33. Preferably, the nucleotide sequence is SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively. Specifically disclosed herein are methylated isolated polynucleotide sequences of MINT3, MINT5, MINT 7, MINTl 1, MINT12, MINTl 3, MINTl 6, MINTl 8, MINT21, MINT25, MINT26, MINT28, and MINT29. Preferably, the nucleotide sequence is SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29, respectively. The term "polynucleotide" or "nucleic acid sequence" refers to a polymeric form of nucleotides at least 10 bases in length. By "isolated polynucleotide" is meant a polynucleotide that is not immediately contiguous with both ofthe coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences. The nucleotides ofthe invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double forms of DNA.
The polynucleotide encoding MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 includes SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, dominant negative forms of MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33, and nucleic acid sequences complementary to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33. A complementary sequence may include an antisense nucleotide. When the sequence is RNA, the deoxynucleotides A, G, C, and T of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 are replaced by ribonucleotides A, G, C, and U, respectively. Also included in the invention are fragments ofthe above-described nucleic acid sequences that are and are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA that encoded by SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 under physiological conditions or a close family member of MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30,
MINT31, MINT32 and MINT33. The term "selectively hybridize" refers to hybridization under moderately or highly stringent conditions which excludes non-related nucleotide sequences. Hybridization conditions have been described above.
The MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINT15, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32, and MINT33 nucleotide sequence includes the disclosed sequence and conservative variations ofthe polypeptides encoded by MINT 1 , MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polynucleotides. The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted arnino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINT15, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 nucleic acid sequences can be expressed in vitro by DNA transfer into a suitable host cell. "Host cells" are cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
In one aspect, the MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31 , MINT32 and MINT33 polynucleotide sequences may be inserted into an expression vector. The term "expression vector" refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation ofthe sequence of interest genetic sequences. Polynucleotide sequence which encode sequence of interest can be operatively linked to expression control sequences.
"Operatively linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. An expression control sequence operatively linked to a coding sequence is ligated such that expression ofthe coding sequence is achieved under conditions compatible with the expression control sequences. As used herein, the term "expression control sequences" refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation ofthe nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to included, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.
By "promoter" is meant minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions ofthe gene. Both constitutive and inducible promoters, are included in the invention (see, e.g., Bitter et al, Methods in Enzymology 1£3_:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage γ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retro virus long terminal repeat; the adeno virus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription ofthe nucleic acid sequences ofthe invention.
In the present invention, the MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINTl 5, MINTl 7, MINT19, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polynucleotide sequence may be inserted into an expression vector which contains a promoter sequence which facilitates the efficient transcription ofthe inserted genetic sequence ofthe host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection ofthe transformed cells. Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg et al. , Gene 56_:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol Chem. 262:3521, 1988) and baculovirus-derived vectors for expression in insect cells. The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g. , T7, metallothionein I, or polyhedron promoters).
MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINT15, MINTl 7, MINT19, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polynucleotide sequences can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences ofthe invention.
By transformation" is meant a genetic change induced in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell). Where the cell is a mammalian cell, the genetic change is generally achieved by introduction ofthe DNA into the genome ofthe cell (i.e., stable).
By "transformed cell" is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding sequence of interest. Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl2 method using procedures well known in the art. Alternatively, MgCl2 or RbCl can be used. Transformation can also be performed after forming a protoplast ofthe host cell if desired.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with DNA sequences encoding the sequence of interest, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors. Cold Spring Harbor Laboratory, Gluzman ed., 1982).
Isolation and purification of microbial expressed polypeptide, or fragments thereof, provided by the invention, may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
In one embodiment, the invention provides substantially purified polypeptide encoded by MINTl, MINT2, MINT4, MINT6. MINT8, MINT 9, MINT10, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 and MINT33 polynucleotide sequences. The term "substantially purified" as used herein refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify a polypeptide encoded by MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTl 0, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31 , MINT32 and MINT33 polynucleotide sequence using standard techniques for protein purification. The substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. The purity of the MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINT10, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31 , MINT32 and MINT33 polypeptide can also be determined by amino-terminal amino acid sequence analysis. Minor modifications ofthe MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32, and MINT33 primary amino acid sequences may result in proteins which have substantially equivalent activity as compared to the unmodified counterpart polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All ofthe polypeptides produced by these modifications are included herein as long as the biological activity still exists.
The polypeptides ofthe invention also include dominant negative forms of the MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 or MINT33 polypeptide which do not have the biological activity of MINTl , MINT2, MINT4,
MINT6, MINT8, MINT 9, MINTIO, MINTl 4, MINTl 5, MINTl 7, MINT19, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 or MINT33 polynucleotide sequence. A "dominant negative form" of MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINT14, MINT15, MINT17, MINTl 9, MTNT20, MINT22, MINT23, MINT24,
MINT27, MINT30, MINT31, MINT32, or MINT33 is a polypeptide that is structurally similar to MINTl, MTNT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 or MINT33 polypeptide but does not have wild-type MINTl , MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 or MINT33 function. For example, a dominant-negative MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 or MINT33 polypeptide may interfere with wild-type MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINTl 4, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24,
MINT27, MINT30, MINT31, MINT32 or MINT33 function by binding to, or otherwise sequestering, regulating agents, such as upstream or downstream components, that normally interact functionally with the MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32 or MINT33 polypeptide.
EXAMPLES
The following examples are intended to illustrate but not to limit the invention in any manner, shape, or form, either explicitly or implicitly. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.
EXAMPLE I DETECTION OF METHYLATED CPG ISLANDS USING MCA
The principle underlying MCA involves amplification of closely spaced methylated Smal sites to enrich for methylated CGIs. The MCA technique is outlined in Figure 1A. About 70 to 80% of CpG islands contain at least two closely spaced (<lkb) Smal sites (CCCGGG). Only those Smal sites within these short distances can be amplified using MCA, ensuring representation ofthe most CpG rich sequences. Briefly, DNA is digested with Smal, which cleaves only unmethylated sites, leaving blunt ends between the C and G. DNA is then digested with the Smal isoschizomer Xmal, which does cleave methylated CCCGGG sites, and which leaves a 4 base overhang. Adaptors are ligated to this overhang, and PCR is performed using primers complementary to these adaptors. The amplified DNA is then spotted on a nylon membrane and can be hybridized with any probe of interest.
As a model experiment, amplification of the pi 6 gene CGI was examined because (1) hypermethylation of this CGI in cancer is well characterized, and correlates with silencing ofthe gene (Herman et al, 1995), and (2) this CGI contains two closely spaced Smal sites (400bp) which can be amplified by MCA. Initially, the reaction was optimized by testing different primers with a variable GC content, and different PCR conditions. As shown in Figure IB, using primers with a 70% GC content, the pi 6 CGI is amplified strongly in the Caco2 cell line, where it is known to be hypermethylated, while no signal above background was detected from any normal colon mucosa. To examine the quantitative aspect of MCA, DNA from Caco2 and normal colon mucosa were mixed in various proportions, and the methylation level of each mix was determined using MCA. MCA detected pi 6 methylation in a semi- quantitative manner between 1% and 100% methylated alleles. Finally, MCA was performed on 109 samples of normal colonic mucosa and adjacent primary colorectal tumor that had previously been typed for pi 6 methylation by Southern blot analysis (Ahuja et al, 1997). MCA and Southern blot were concordant in 107/109 (98%) ofthe cases. In one case, MCA detected a low level of methylation (5-10 %) in a cancer sample that had been judged negative by Southern blot. In the other discordant case (positive by MCA, negative by Southern blot), the discordance may be related to heterogeneous pi 6 methylation, as has been described (Costello et al, 1996). MCA is a novel PCR-based technique that allows for the rapid enrichment of hypermethylated CG rich sequences, with a high representation of methylated CpG islands. This technique can have several potential applications. MCA is very useful for the determination ofthe methylation status of a large number of samples at multiple loci simultaneously. By optimizing the PCR conditions, it should be readily adaptable to the study of the methylation status of any gene that has two closely spaced Smal sites. As shown herein, there is a very high concordance rate between MCA and other methods for the detection of hypermethylation such as Southern blot analysis and bisulfite-based methods. However, MCA (1) requires good quality DNA, excluding the study of paraffin-embedded samples, (2) examines only a limited number of CpG sites within a CGI and (3) is sensitive to incomplete digestion using the methylation-sensitive enzyme Smal. Nevertheless, many steps in MCA are amenable to automation and, by allowing for the examination of multiple genes relatively quickly, may have important applications in population-based studies of CGI methylation.
EXAMPLE 2 IDENTIFICATION OF DIFFERENTIALLY METHYLATED CG IN CRC BY MCA/ RDA
To identify novel CGIs abeπantly methylated in CRC, RDA (Lisitsyn et al, 1993) was performed on MCA amplicons from the colon cancer cell line Caco2 as a tester, and a mixture of DNA from the normal colon mucosa of 5 different men (to avoid cloning polymorphic Smal sites or inactive and methylated X chromosome genes from women) as a driver. Two separate experiments were conducted, one using a lower annealing temperature (72/C), and the other using a higher annealing temperature (77/C) and more GC rich primers. After two rounds of RDA, the PCR products were cloned, and colonies containing inserts were identified by PCR. Based on initial experiments, we expected most ofthe recovered clones to contain Alu repetitive sequences, which are CG rich and hypermethylated (Kochanek et al, 1993). All clones were therefore probed with an Alu fragment, and only non-hybridizing clones were analyzed further. Out of 160 non-Alu clones, 46 were independent clones and 33 of these (MINTl -33, Methylated in .Tumors, SEQ ID NOs:l-33, respectively) appeared to be differentially methylated in Caco2 cells by comparing hybridization to MCA products from Caco2 and normal colon (Figure 1C). 19 ofthe clones (MINTl -19) were obtained using the lower annealing temperature, and 14 (MINT 20-33) using the higher temperature.
To confirm the aberrant methylation of these clones, Southern blot analysis was performed using DNA digested with Smal or Xmal. All ofthe 33 clones were hypermethylated in Caco2 compared to normal colon mucosa. Of these 33, one clone (MINTl 3) detected highly repeated sequences and two clones (MINTl 8 and MINT28) appeared to correspond to mildly repeated gene families (data not shown). All others appeared to detect single copy DNA fragments. In addition, hypermethylation at CpG sites within the clones and distinct from the Smal sites was confirmed by bisulfite-PCR for 6 clones. In each case, Caco2 was found to be hypermethylated at these sites.
By DNA sequencing (example shown in Figure 2), we found that 29 clones had a GC content greater than 50%, and satisfied the minimal criteria for CGIs (200bp, GC content>50%, CpG/GpO0.5) (Gardiner-Garden and Frommer, 1987). As might be expected, clones obtained with the higher annealing temperature and more GC rich primers had a relatively higher GC content (Table 1). The size of each clone, percentage of GC nucleotide, observed expected CGs, sequence homology and, chromosomal location are summarized in Table 1. MINT5, MINT8, MINTl 1, MINT14 and MINT16 contained GC rich regions only in one end ofthe clones, and these may have been recovered from the edge of CGIs.
Table 1: Summary ofthe 33 Differentially Methylated Clones Isolated by MCA-RDA
Clone Size %GC O/E CGI Blast Homology Chromosome Methyla(bp) Map tion Pattern
MINTl 528 56 0.6 Yes None 5ql3-14 Type C
MINT2 562 50 0.8 Yes None 2p22-21 Type C
MINT3 563 55 1 Yes Human EST AA557808 lp34-35 Type a
MINT4 481 60 0.8 Yes None 15q25-26 Type a
MINT5 852 46 0.5 Yes* Human CpG clone 88c 1 14q21-22 Type a
MINT6 401 59 0.6 Yes None 12ql4-15 Type a
MINT7 481 49 0.9 Yes Human genomic DNA 6p21-22 Type A
MINT8 617 46 0.5 Yes* None N.D Type A
MINT9 605 54 0.3 No None lp34-35 Type A
MINTIO 608 49 0.6 Yes None 9q34-Ter Type A
MINTl 1 637 49 0.6 Yes* Versican 5ql2-13 Type A
MINT12 552 49 0.6 Yes CpG clone 33h2 7q31-32 Type C
MINTl 3 308 60 0.9 Yes LINE1 N.D Cell line
MINTl 4 620 54 0.4 Yes* None 10pl3-15 Type A
MINTl 5 641 53 0.7 Yes None l lpl2-13 Type A
MINT16 664 62 0.5 Yes* Alpha-tubulin 2q Type A
MINTl 7 491 54 0.7 Yes None 6 Type C
MINTl 8 435 58 0.1 No Acrogranin N.D Cell line
MINT 19 443 55 0.2 No None N.D Type A
MINT20 510 67 0.8 Yes mouse OTP N.D Type A
MINT21 411 62 0.4 No None 22ql3 Type A
MINT22 438 60 0.9 Yes None 10pl2 Type A
MINT23 346 64 0.8 Yes Csx 5q34-35 Type A
MINT24 525 63 0.7 Yes None 3p25-26 Type A
MINT25 339 60 0.7 Yes Human genomic DNA 22ql l Type C
MINT26 591 58 0.8 Yes CpG clone 73el 7ql l Type A
MINT27 242 74 0.7 Yes None N.D Type C
MINT28 463 58 1 Yes Ribosomal RNA gene N.D Type A
MINT29 429 60 0.7 Yes CpG clone 20b 1 7ql l N.D
MINT30 536 65 0.5 Yes None 20ql l Type A
MINT31 673 65 0.8 Yes None 17q21 Type C
MINT32 464 66 1 Yes None 20ql3 Type A
MINT33 139 65 0.8 Yes None N.D N.D
O/E : Observed expected numbers of CpGs. N.D : not determined. * Only one portion ofthe clones has a CpG island. By DNA homology search using the BLAST program (BLAST 2.0, default parameters, see http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph- newblast?J-form=0), 4 clones were identical to human gene sequences, four clones were identical to CGIs randomly sequenced from a CGI library (Cross et al, 1994), one was identical to an EST, two clones were identical to high throughput genomic sequences deposited in Genbank, three clones had significant homology to other genes and the other 19 had no significant match in the database; MINTl 1 was identical to exon 1 and intron 1 ofthe human versican gene (Zimmerman et al, 1989), and corresponded to the 3' edge of a promoter associated CGI; MINT14 was identical to exon 1 ofthe human alpha-tubulin gene (Dobner, P.R., et al, 1987), and was also the 3' edge ofthe CGI; MINT 24 coπesponded to the 3' noncoding region ofthe human homeobox gene CS (Turbay et al, 1996); MINT21 had a region with 94% homology at the nucleotide level to exon 2 ofthe mouse OPT gene (Simeone et al., l 994) and probably represents the human homologue of this gene; MINT28 was homologous to ribosomal gene sequences; MINTl 8 was homologous to the acrogranin gene family. To examine the presence of potential promoter sequences in these clones, promoter prediction was performed using several computer programs (see programs available at http://dot.imgen.bcm.tmc.edu:9331/seq-search/gene-search.html). Twenty out ofthe 33 clones were predicted as promoters using the NNPP program, and 6 were predicted as promoters by using the TSSG program.
The chromosomal position of most ofthe unknown clones was determined using a somatic cell hybrid panel and a radiation hybrid panel (Table 1). Of note, MINT3 and MINT9 mapped to chromosome lp35-36, MINT 13 mapped to 7q31, MINT24 mapped to 3p25-26, MINT25 mapped to 22ql 1-Ter, and MINT31 mapped to 17q21. All of these chromosomal segments are areas that are frequently deleted in various tumors.
An important application of MCA is in the discovery of novel genes hypermethylated in cancer. As demonstrated here, MCA coupled with RDA is a rapid and powerful technology for this purpose, and compares favorably with other described techniques (Hayashizaki et al, 1994, Gonzalgo et al, 1997; Huang et al, 1997). In addition to the identification of genes hypermethylated in cancer, MCA could potentially be used to discover novel imprinted genes using parthenogenetic DNA (Kaneko-Ishino et al. , 1995), as well as novel X-chromosome genes.
EXAMPLE 3 SILENCING OF THE VERSTCAN GΕME IN CRC
To determine whether some of these clones truly represented genes silenced by methylation, we examined the versican gene in more detail. Versican is a secreted glycoprotein that appears to be regulated by the Rb tumor suppressor gene (Rohde et al, 1996). MINTl 1 corresponds to part of exon 1 and part of intron 1 ofthe versican gene (Figure 3 A). Hypermethylation ofthe two Smal sites in exon 1 and intron 1 in colon cancer cell lines was confirmed by both Southern blot analysis and MCA. In order to determine if this methylation was representative ofthe entire CGI, including the proximal promoter, PCR was performed on bisulfite-treated DNA using primers designed to amplify the region around the transcription start site of this gene. The PCR product was then digested with restriction enzymes that distinguish methylated from unmethylated DNA. The versican promoter was found to be completely methylated in the colon cancer cell lines, DLD1, LOVO, SW48 and SW837, and partially methylated in HCT116 and HT29 (Figure 3B). In primary colon tumors, versican was hypermethylated in 17 out of 25 cases (68%). Interestingly, some methylation ofthe versican promoter was also found in normal tissues, albeit at lower levels when compared to tumors. The level of methylation in normal colon mucosa increased with age ofthe patient (Fig3C), from an average of 6.9% in patients between 20 and 30 years of age, to an average of 28.9% in patients over 80. A linear regression analysis revealed a significant association between age and versican promoter methylation (R=0.7, P<0.000001). Using RT-PCR, we next examined the expression of versican in normal colon mucosa and CRC cell lines. Versican was found to be expressed in normal colon epithelium, but was markedly down-regulated or absent in methylated colon cancer cell lines. Expression of versican in all these cell lines was easily restored after treatment with the demethylating agent, 5-aza-deoxycytidine. These data suggest that versican becomes methylated in normal colon in an age-dependent manner, and that this leads to hypermethylation and loss of expression in most colorectal tumors.
Using MCA/RDA 33 differentially methylated clones were identified and characterized in detail. By sequencing, we found that 29 out ofthe 33 clones satisfy the criteria of CpG islands, demonstrating that MCA can represent CGIs specifically. Of these 29 clones 5 were already known genes (versican, alpha-tubulin, CSX, OPT homologue and ribosomal RNA gene). Of these, versican is most interesting in that this proteoglycan is an Rb inducible gene (Rohde et al. , 1996), suggesting that down regulation of this gene product may have an important role in colorectal carcinogenesis, where Rb mutations are rare. The data clearly show that aberrant methylation ofthe versican gene promoter is correlated with silencing of this gene. In addition, methylation ofthe alpha-tubulin gene in Caco2 is consistent with the results of studying the gene expression profile of colorectal cancers using SAGE (Zang et al, 1997), which demonstrated that alpha-tubulin is markedly down-regulated in CRC. Methylation ofthe CSX and OPT genes does not coincide with their 5' end, and is therefore not expected to silence these genes. It is possible, however, that these CpG islands are associated with alternate transcripts ofthe genes, or with other nearby genes, which would then be silenced by methylation (Wutz, A., et al, 1997). Finally, methylation of ribosomal genes has previously been seen in aging tissues (Swisshelm, K., et al, 1990) and therefore is not surprising to find in cancers. Because some of the clones recovered are in the exon 1 region of expressed genes, identification of new tumor suppressor genes might be facilitated by using MCA RDA clones as probes for screening cDNA library. Indeed, based on their chromosome location, several clones map to chromosomal regions thought to harbor TSGs because they are highly deleted in various tumors (e.g. , chromosome lp35, 3p25-26, 7q31, 17q21 and 22ql l-Ter).
EXAMPLE 4 TWO TYPES OF METHYLATION IN CRC By examining the methylation status of several known genes in colorectal tumors, it has been previously demonstrated that some genes tend to be methylated in an age-dependent manner in normal colon (Issa et al, 1994), and are frequently methylated in CRC, while others are methylated in cancers exclusively (Ahuja et al, 1997). To examine this issue on a genome wide level in some detail, the methylation profile of 31 MINT clones in a panel of colorectal tumors and corresponding normal colon mucosa was examined using MCA (two clones could not be accurately studied because of high background (MINT29) or small size (MINT33)). Because all of theses clones were recovered from a CRC cell line, there was an initial concern that many of these were not representative of methylation in primary (uncultured) tumors. However, of the 31 clones, 29 were also found to be methylated in some primary CRC. The two clones methylated only in the cell line Caco2 were (1) MINT14, a LINE element, and (2) MINTl 8, a sequence that had a very low CpG frequency and did not qualify as a CGI. Thus, all non-repetitive CGIs recovered were methylated in primary CRC as well as cell lines. Hypermethylation patterns of these 29 clones fell into two distinct categories. A majority ofthe clones (22 out of 29) were found to be frequently methylated (>70%) in the tumors tested, and a slight amount of methylation was also detected in normal colon mucosa. For all of these clones, the normal colon mucosa obtained from young patients showed less methylation compared to the normal mucosa from older patients (Figure 4B). Thus, the majority of CGIs hypermethylated in CRC are methylated in normal colon mucosa as well, in an age related manner. This methylation was named Type A for aging-specific methylation.
The remaining 7 clones were methylated exclusively in CRC, and their frequency of methylation was significantly lower than type A methylation (ranging from 10% to 50%). This type of methylation was named type C for cancer-specific.
Recently, several reports have suggested that aberrant methylation of CGIs may play an important role in cancer development (Baylin et al, 1998; Jones 1997). However, there is little integrated information on aberrant CGI methylation in cancer at multiple loci, probably because ofthe lack of a method to detect methylation in a large number of samples for unselected CGIs throughout the genome. Furthermore, it has been shown that cultured cell lines have a high degree of CGI methylation (Antequera et al, 1990) but it was not known to what extent this reflects methylation in primary cancers. To address these issues, the relatively quantitative and high output features of MCA allowed us to determine the methylation profile of 31 differentially methylated loci in a panel of colorectal carcinomas.
Despite the fact that all sequences were initially recovered from a colon cancer cell line, only 2 out ofthe 31 clones showed cell line restricted methylation. From the sequence data, one of these two clones was a repeated sequences (LINEl), and the other was not a CGI. Thus most ofthe single copy clones recovered proved to be methylated not only in cell lines but also in some primary colon cancers. Analysis of these 29 clones revealed two distinct types of hypermethylation in cancer (Type A for aging and Type C for cancer), which may have distinct causes, and different roles in cancer development. Type A methylation was seen in the majority of these clones: 22 of 29 (74%) clones were methylated in an age-related manner in normal colon tissue, and hypermethylated at a high frequency in CRC, as we have shown for the ER gene (Issa et al, 1994) and others (Issa et al, 1996; Ahuja et al. , submitted). These results suggest that a large number of CGIs in the human genome are incrementally methylated during the aging process and, for many genes, this methylation correlates with reduced gene expression as shown for ER (Issa et al. , 1994) and versican. Although the mechanism of Type A methylation is unknown, it is likely to result from physiological processes rather than a genetic alteration because (1) it is very frequent and affects large numbers of cells, (2) it is present in all individuals, not just patients with cancer and (3) this process is gene and tissue specific (Ahuja et al, submitted). Because the methylation status at a given CGI is thought to be related to positive (methylator) factors (Mummaneni et al, 1993; Mummaneni et al, 1995; Magewu and Jones, 1994, Nertino et al, 1996) and negative (protector) factors (Macleod et al, 1994; Brandeis et al, 1994; Turker and Bestor, 1997; Chen et al, 1997), it is possible that for some genes, this balance favors slightly de-novo methylation, and that this is reflected by progressive hypermethylation after repeated cell divisions.
EXAMPLE 5 GLOBAL HYPERMETHYLATION IN CRC
To understand the patterns of cancer-specific methylation in CRC, the methylation status of all 7 type C clones was analyzed, as well as pi 6 in primary cancers and polyps (Figure 4). Two of these clones (MINTl and MINT2) were studied by both MCA and bisulfite-PCR, and the concordance between the two techniques was found to be 98%. PI 6 was studied by both MCA and Southern blot, with a concordance rate of 98%. When we considered the six clones that were methylated in more than 10% ofthe cases, as well as pi 6, a remarkable pattern emerged (summarized in Figure 5 and Table 2). Table 2: Percentage of Tumors Methylated at Multiple MINT Loci, p!6 and hMLHI.
Type C Methylation Type A Methylation
MINT MINT MINT MINT MINT MINT pl6 hMLH MINT MINT MINT MINT MINT MINT 1 2 12 17 25 31 1 6 8 22 24 30 32
MI+ Cancer EI+ (n=12) 100% 100% 100% 92% 100% 92% 83% 100% 100% 100% 100% 100% 100% 100% ι El- (n=4) 25% 0% 0% 0% 0% 0% 0% 25% 100% 100% 100% 75% 75% 50%
MI- Cancer EI+ (n=17) 65% 82% 59% 41% 100% 82% 59% 0% 94% 100% 100% 100% 60% 93% liI- (n-= ! 7) 0% 12% 0% 0% 12% 0% 0% 0% 82% 86% 100% 82% 40% 65%
Adenoma EI+ (n=7) 86% 86% 71% 29% 86% 71% 57% 0% 86% 83% 100% 100% 43% 86% El- (n=8) 0% 0% 0% 0% 0% 13% 0% 0% 50% 100% 86% 50% 13% 25%
Figure imgf000054_0001
The 50 CRC fell into two distinct groups: (1) A group with a high level of Type C methylation, whereby all the tumors had methylation of 4 or more loci simultaneously and (2) a group where methylation of any type C clone is extremely rare. Thus, the first group of tumors appears to display profound global hypermethylation (GH+), which is lacking in the second group (GH-). Interestingly, there was a great concordance between methylation of the pi 6 gene, which was not selected for by our cloning process, and the presence of GH. In sharp contrast, Type A methylation was not significantly different between GH+ and GH- tumors (Table 2).
GH was also detected in a subset of colorectal adenomas (Figure 5), suggesting that it is an early event in carcinogenesis. Interestingly, while 5 of 5 small adenomas (<7 mm) were GH-, 6 of 9 large adenomas (>10 mm) were GH+, suggesting that this defect may be acquired in the transition between small and large adenomas. In 6 cases, both an adenoma and a cancer from the same patients were examined. In one of these, GH was detected both in the adenoma and the cancer; in 3 cases, GH was detected in the cancer but not in the adenoma and in 2 cases, GH was detected in neither the adenoma nor the cancer.
By contrast to type A methylation, type C methylation is relatively infrequent in primary CRC, and is never observed in normal colon mucosa. Furthermore, detailed analysis of type C methylation in CRC revealed a striking pattern, suggesting the presence of global hypermethylation in a subset of these tumors: GH positive cases are characterized by frequent and concordant methylation of all type C clones examined, such that each tumor has at least four methylation events. By contrast, type C methylation is virtually non-existent in tumors without GH. This concordance cannot be due to simple experimental variation or artifacts because (1) methylation was verified using separate methods (MCA, bisulfite-PCR and Southern blots), (2) the concordance was not limited to MCA/RDA derived clones since it also affected the pl6 (Herman et al, 1995) and hMLHI (Kane et al, 1997) genes, and (3) there was no significant difference in type A methylation between GH+ and GH- tumors. Global hypermethylation appears to be an early event in the development of CRC, being detectable in large pre-neoplastic adenomas. Because many genes are potential candidates for inactivation through promoter methylation (Baylin et al. , 1998; Jones, 1996), global hypermethylation may have profound pathophysiologic consequences in neoplasia through the simultaneous inactivation of tumor-suppressor genes (such as pi 6), metastasis-suppressor genes (such as E-cadherin), angiogenesis inhibitors (such as Thrombospondin-1) and others. In fact, our data suggest that global hypermethylation could also result in mismatch repair deficiency through methylation and inactivation ofthe hMLHI promoter, and may explain up to 75% of cases of sporadic CRC with microsatellite instability. The causes of type A and type C methylation are probably different because the latter is detected only in a limited number of cases, and the genes affected are different. Because ofthe remarkable concordance in type C methylation among GH+ cases, it appears likely that these tumors all share a specific defect in the maintenance ofthe methylation-free state in CGIs. This defect could be either abeπant de-novo methylation (through a mutation in DNA-methyltransferase for example), or loss of protection against de-novo methylation, through the loss of a trans-activating factor (Macleod et al. , 1994; Chen et al, 1997). Because DNA-methyltransferase activity is similar in the two groups, the latter hypothesis is more likely. Thus, at least in colorectal cancer, it appears likely that type C methylation (an epigenetic error) is actually caused by a genetic event that results in an increased chance of methylating a subset of CGIs. Ironically, this epigenetic defect may then result in additional genetic lesions through the induction of mismatch-repair deficiency.
EXAMPLE 6
MICROSATELLITE INSTABILITY IS LINKED TO
GLOBAL HYPERMETHYLATION IN CRC In a previous study (Ahuja et al. , 1997), a link was reported between microsatellite instability and a hypermethylator phenotype in sporadic CRC. Relatively few mutations in mismatch repair genes have been reported in sporadic MI+ cancers, but hMLHI methylation has recently been observed in some cases (Kane et al, 1997). To determine the relation between global hypermethylation and microsatellite instability in CRC, we measured hMLHI methylation using bisulfite/PCR in our panel of CRC which had also been previously typed for the presence of microsatellite instability (Figure 5). hMLHI was studied by bisulfite-PCR only because it does not have 2 Smal sites in its CGI. Overall, 16 out of 50 (32%) cancers had evidence of microsatellite instability. Among the 29 GH+ cases, 12 had evidence of hMLHI methylation, suggesting that hMLHI is one ofthe targets of global hypermethylation in CRC. All of these 12 tumors had microsatellite instability. By contrast, hMLHI methylation was detected in only one ofthe 21 GH- cases. These data establish a strong link between the GH phenotype, hMLHI methylation and microsatellite instability in CRC. Two lines of evidence suggest that microsatellite instability may follow, and be caused by, global hypermethylation and hMLHI methylation. First. GH is detectable in about half of colonic adenomas, but none of these tumors have hMLHI methylation, and microsatellite instability is extremely rare in this pre- neoplastic lesion (Samowitz and Slattery, 1997). Second, GH is not simply caused by mismatch repair defects because microsatellite instability is absent in more than half of the GH+ cases, and GH was absent in 4 of the 16 cancers with microsatellite instability. Overall, our data suggest that, in sporadic CRC, the majority (12 out of 16, or 75%) of cases with microsatellite instability may be caused by GH followed by hMLHI methylation, loss of hMLHI expression and resultant mismatch repair deficiency (Herman et al. , submitted).
Based on these data, the following model has been developed integrating CGI methylation into CRC development (Figure 6). In this model, CGI methylation plays two distinct roles, and appears to arise through distinct mechanisms. Initially, type A methylation arises as a function of age in normal colorectal epithelial cells. By affecting genes that regulate the growth and/or differentiation of these cells, such methylation results in a hyperproliferative state, which is thought to precede tumor formation in the colon (reviewed by Lipkin, 1988). Such hyperproliferation is known to arise with age in colorectal epithelium (Holt et al, 1988, Roncucci et al, 1988), and to be marked in patients with CRC. The cause of type A methylation is unknown, but without being bound by theory it is possible that it is related to endogenous factors inherent to the structure of DNA, and that it may be modulated by factors such as level of ongoing expression and exposure to carcinogenic insults. Furthermore, modulation of type A methylation may provide one possible explanation for the reduction in CRC tumorigenesis by reducing levels of DNA-methyltransferase (Laird et al, 1995). A second major role for CGI methylation appears later, perhaps at the transition between small and large adenomas in the colon. This methylation (type C) affects only a subset of tumors, which then evolve along a pathway of global hypermethylation. This GH leads to cancer development through the simultaneous inactivation of multiple tumor-suppressor genes such as p 16, and induction of mismatch repair deficiency through inactivation of hMLHI . The cause of this global hypermethylation is unknown, but may well be related to inactivation of a gene that protects CGIs from de-novo methylation. Finally, we propose that tumors without GH evolve along more classic genetic instability pathways, including chromosomal instability (Lengauer et al. , 1997 A). Interestingly in this regard, Lengauer et al found an inverse coπelation between chromosomal instability and MMR deficiency in CRC cell lines (Lengauer et al, 1997B).
While based on CRC, this model is applicable to most human malignancies. In evidence has also been found for type A and type C methylation in brain tumors (Li et al, 1998). Preliminary evidence also suggests the presence of global hypermethylation in multiple types of cancers, including stomach cancers, brain tumors and hematopoietic malignancies.
In conclusion, a novel method, MCA, has been developed to selectively amplify methylated CGIs. Using MCA/RDA 33 differentially methylated clones in CRC were isolated. The methylation profile of these clones revealed that nearly all methylation in CRC can be accounted for by (1) age-related methylation and (2) a hypermethylator phenotype presumably caused by global hypermethylation. Deciphering the mechanisms underlying these phenomena should facilitate the early detection, prevention and therapy of cancers, including colorectal cancers. EXAMPLE 7
IDENTIFICATION OF CACNA 1G AS A TARGET FOR
HYPERMETHYLATION ON HUMAN CHROMOSOME I7q21
To identify genes differentially methylated in colorectal cancer, methylated CpG island amplification was used followed by representational difference analysis (Razin and Cedar, Cell 17: 473-476, 1994, herein incorporated by reference). One ofthe clones recovered (MINT31, see above) mapped to human chromosome 17q21 using a radiation hybrid panel, and a Blast search revealed this fragment to be completely identical to part of a BAC clone (Genbank: AC004590) sequenced by high throughput genomic sequence. The region suπounding MINT31 fulfills the criteria of a CpG island: GC content 0.67, CpG/GpC ratio 0.78 and a total of 305 CpG sites in a 4 kb region. Using this CpG island and 10 kb of flanking sequences in a Blast analysis, several regions highly homologous to the rat T-type calcium channel gene, CACNA1G, were identified (Perez-Reyes et al., Nature 29J.: 896-900. 1998, herein incorporated by reference). Several ESTs were also identified in this region. Using Genscan, 2 putative coding sequences (Gl, and G2) were identified. Blastp analysis revealed that Gl has a high homology to the EH- domain-binding protein, epsin, while G2 is homologous to a C-elegans hypothetical protein (accession No. 2496828).
The MINT31 CpG island corresponds to the 3' regions of Gl and G2, based on the direction of the open reading frame and the presence of a poly A tail, and is unlikely to influence their transcription. The EST closest to MINT31 (HI 3333) was sequenced entirely and was found not to contain a continuous open reading frame, but a poly-adenylation signal was identified on one end, along with a poly A tail. These data suggest that HI 3333 coπesponds to the last 2 exons of an unidentified gene. MINT31 is in the intron of this gene and is unlikely to influence its transcription. However, based on both promoter prediction (TSSG) analysis of this region and homology to the rat CACNAIG sequence, the MINT31 CpG island is also in the 5' region of human CACNAIG gene and may play a role in its transcriptional activity.
The human CACNAIG sequence deposited in Genbank lacks the 5' region ofthe gene, when compared to the rat homologue. To determine the 5' region of human CACNAIG, we amplified cDNA by RT-PCR using primers based on the BAC sequence (Genbank: AC004590, herein incorporated by reference). The PCR products were cloned and sequenced, and the genomic organization ofthe gene was determined by comparing the newly identified sequences as well as the known sequences to the BAC that covers this region. CACNAIG is composed of 34 exons which span a 70 kb area. Based on sequences deposited in Genbank, the gene has two possible 3' ends caused by alternate splicing. CACNAIG is highly homologous to rat CACNAIG with 93% identity at the protein level, and 89% identity at the nucleotide level. The 5' flanking region of CACNAIG lacks TATA and CAAT boxes, which is similar to many housekeeping genes. A putative TFIID binding site was identified 547-556 bp upstream from the translation start site, and several other potential transcription factor binding sites such as API (1 site), AP2 (2 sites) and SP1 (10 sites), were identified upstream of CACNAIG exon 1 using the promoter prediction program, TESS (data not shown).
The CACNAIG CpG island is 4 kb, and is larger than many typical CpG islands. MINT31 corresponds to the 5' edge ofthe island while CACNA1G is in the 3' region. It is not known whether large CpG islands such as this are coordinately regulated with regards to protection from methylation, and aberrant methylation in cancer. To address this issue, the methylation status ofthe 5' region of CACNAIG was studied using bisulfite-PCR of DNA from normal tissues as well as 35 human cancer cell lines from colon, lung, prostate, breast and hematopoietic tumors. The CpG island was divided into 8 regions and their methylation status was examined separately. The genomic DNA was treated with sodium bisulfite and PCR amplified using primers containing no or a minimum number of CpG sites. Methylated alleles were detected by digesting the PCR products using restriction enzymes which specifically cleave sites created or retained due to the presence of methylated CpGs. None ofthe regions was methylated in normal colon, consistent with a uniform protection against de-novo methylation.
Regions 1 and 2 were frequently methylated in cancer cell lines, and behaved in a concordant manner. These 2 regions were methylated in most cancer cell types except gliomas, and most cell lines where methylation was found methylated both regions simultaneously. Region 3, which is less CG rich than any ofthe other regions, had either no methylation or very low levels of methylation in most cell lines. Regions 5, 6, and 7 behaved quite differently compared to 1-3. Methylation of these regions was less frequent than regions 1-2, as 22/35 cell lines had no detectable methylation there, despite often showing methylation of region 1-2. However, when methylation was present (in 13/35 cell lines), it affected all 3 regions simultaneously, although to varying extents. Finally, regions 4 and 8 behaved differentially again, being partially methylated primarily in colon and breast cell lines. Therefore, with regards to hypermethylation in cancer, the CpG rich region upstream of CACNAIG appears to be composed of 2 CpG islands which behave independently. MINT31 corresponds to the upstream CpG island (island 1, regions 1 and 2), while the 5' region of CACNAIG is contained in the downstream CpG island (island 2, regions 5-7). Regions 3, 4 and 8 correspond to the edge of these CpG islands, and behave a little differentially than the hearts ofthe CpG islands, as previously described for the E-Cad gene (Graff, et al., J. Biol. Chem. 221: 22322-22329, 1997).
Overall, the methylation patterns fell into 5 distinct categories: (1) No methylation in any region (normal tissue). (2) Slight methylation of island 1 (6 cell lines, see for example TSU-PRL in Fig. 2). (3) Heavy methylation of island 1 but no methylation of island 2 (16 cell lines, see for example Caco2 in Fig. 2). (4) Heavy methylation of island 1 and moderate to heavy methylation of island 2 (6 cell lines, see for example RKO and Raji in Fig. 2). (5) High methylation of island 1 and low to moderate methylation of island 2 (7 cell lines, see for example MB-231 in Fig. 2).
In a previous study of rat CACNAIG, this gene was shown to be expressed most abundantly in the brain (Perez-Reyes et al., Nature 391 : 896- 900. 1998). To determine the expression of CACNAIG in normal and neoplastic human cells, RT-PCR was performed using cDNA from various normal tissues and from a panel of 27 tumor cell lines. CACNAIG was expressed ubiquitously in a variety of tissues and cell lines. In normal tissues expression was relatively low but easily detectable, while most cell lines had relatively high expression of CACNAIG. However, some cell lines had negligible or totally absent levels of CACNAIG expression. The results of CACNAIG expression was coπelated with the detailed methylation analysis previously described. In this analysis, a remarkable pattern emerged. Methylation of region 1-4 and 8 had no effect on CACNAIG expression. However, there was a strong correlation between methylation of regions 5-7 and expression ofthe gene. In fact, all cell lines that lack methylation of this region strongly express the gene. All 6 cell lines with pattern 4 methylation studied had no detectable expression. Finally, the 7 cell lines with pattern 5 methylation (examples DLD-1 and MB-453) had variable levels of expression ranging from very low to near normal. The fact that patterns 3 and 5 differ significantly with regards to expression, but are almost identical with regards to methylation of all regions except 7 suggests that this area is important in the inactivation of CACNAIG.
To confirm whether methylation ofthe 5' CpG island of CACNAIG is really associated with gene inactivation, 3 non-expressing cell lines showing pattern 4 methylation (RKO, SW48 and Raji) and 2 weakly expressing cell lines showing pattern 5 methylation (MB-231 and MB-435) were treated with 1 M of the methyl-transferase inhibitor 5-deoxy-azacitidine. After treatment, all these cell lines re-expressed CACNAIG mRNA. Consistent with re- expression, demethylation of region 7 was observed after 5-deoxy-azacitidine treatment (Fig. 3C).
De novo cytosine methylation is thought to sometimes occur in vitro during cell propagation (Antequera et al., Cell 6.2: 503-514, 1990). To determine whether the methylation of CACNAIG occurs in vivo, primary human tumors were examined for methylation ofthe 5' region of CACNAIG. Aberrant methylation was detected in 17 out of 49 (35%) colorectal cancers, 4 out of 28 colorectal adenomas (25%), 4 out of 16 (25%) gastric cancers and 3 out of 17 (18%) acute myelogenous leukemia cases. In colorectal cancers, there was a significant correlation between methylation of CACNAIG and methylation of pi 6 (p<0.005) and hMLHI (pθ.001), as well as a strong correlation with the presence of microsatellite instability, and the recently identified CpG island methylator phenotype (CIMP), supporting that CACNAIG is also a target for CIMP in colorectal cancer.
To determine whether aberrant methylation ofthe 5' region of
CACNAIG affects the expression status of this gene in primary tumors, we performed RT-PCR using cDNA from a series of colorectal adenomas. Six out of 8 cases which showed no methylation of region 7 expressed CACNAIG. In sharp contrast, all 5 cases that showed methylation of region 7 had no detectable expression of this gene.
Thus, a human T-type calcium channel gene (CACNAIG) has been identified and cloned using the MINT31 sequence as a probe. The human T- type calcium channel gene has been determined to be a target of aberrant methylation and silencing in human tumors. The data show that MINT31 (a representative sequence of MINT 1-33) can be used as a probe to identify genes that play a role in disorders such as cell proliferative disorders.
Detailed analysis ofthe CpG island upstream of CACNAIG revealed that methylation 300 to 800 bp upstream ofthe gene closely coπelated with transcriptional inactivation. The CACNAIG promoter is contained in a large CG rich area that is not coordinately methylated in cancer. The CpG island around MINT31 is much more frequently methylated in cancers compared to that just upstream of CACNAIG. This may simply be caused by differential susceptibility to de-novo methylation between these two regions, with methylation of MINT31 serving as a trigger, and eventually spreading to CACNAIG, as described in other genes (Graff, et al., J Biol. Chem. 272: 22322-22329, 1997). However, it is likely that these 2 regions are controlled by different mechanisms because (1) cell lines kept in culture for countless generations do not in fact spread methylation from MINT31 to CACNAIG (e.g., Caco2), (2) region 3 that separates the 2 islands is infrequently and sparsely methylated in cancer and (3) 2 cases of primary colorectal cancer were found which are methylated at the CACNAIG promoter but not at
MINT31). Therefore, methylation of MINT31 appears to be independent of methylation of CACNAIG suggesting that they are 2 distinct CpG islands regulated by different mechanisms. These data leave open the possibility that MINT31 is the promoter for an unidentified gene, which may perhaps be transcribed opposite to CACNAIG.
Many CpG islands of silenced genes appear to be methylated uniformly and heavily throughout the island (e.g., Graff, et al., J. Biol. Chem. 272: 22322-22329, 1997). In contrast the methylation patterns ofthe 5' region of CACNAIG (region 5-7) was heterogeneous in the cell lines which did not express this gene. Nevertheless, methylation does appear to play a role in CACNAIG repression since demethylation readily reactivates the gene.
The causes of CACNAIG methylation remain to be determined.
Methylation was not detected in normal colon mucosa, placenta, normal breast epithelium and normal bone marrow, including samples from aged patients, suggesting that methylation of this region is cancer specific. However, there was a significant correlation between methylation of CACNAIG and other tumor suppressor genes such asp 16 and hMLHI. Thus, CACNA 1 G probably is a target for the recently described CIMP phenotype, which results in a form of epigenetic instability with simultaneous inactivation of multiple genes. It should be noted that a gene identified by the method ofthe invention (MINT31) has been successfully utilized to identify another gene of interest (CACNAIG) whose methylation pattern coπelates with the presence of specific cell proliferative disorders.
T-type calcium channels are involved not only in electrophysiological rhythm generation but also in the control of cytosolic calcium during cell proliferation and cell death (reviewed in Berridge, et al., Nature 395: 645- 648, 1998). The results demonstrate that the expression of CACNAIG is not limited to brain and heart, suggesting that it may play a role in these other tissues. It has previously been shown that Ca2+ influx via T-type channels is an important factor during the initial stages of cell death such as apoptosis (Berridge, et al., Nature 2 1: 645-648, 1998), ischemia (Fern, J. Neurosci. 18: 7232-7243, 1998) and complement-induced cytotoxicity (Newsholme, et al., Biochem. J. 221 773-779, 1993.). These studies determining the methylation status of the CACNAIG suggest that the impairment of voltage gated calcium channels may play an important role in cancer development and progression through altering calcium signaling.
EXAMPLE 8 EXPERIMENTAL PROCEDURES
Methylated CpG Island Amplification.
The procedure is outlined in Figure 1. Five μg of DNA were digested with 100 units of Smal for 6 hours (all restriction enzymes were from NEB). The DNA was then digested with 20 units of Xmal for 16 hours. DNA fragments were then precipitated with ethanol. RXMA and RMCA PCR adaptors were prepared by incubation ofthe oligonucleotides RXMA24 (5'-AGCACTCTCCAGCCTCTCACCGAC-3') (SEQ ID NO: 34) and RXMA12 (5'-CCGGGTCGGTGA-3') (SEQ ID NO:35), or RMCA24 (5'-CCACCGCCATCCGAGCCTTTCTGC-3') (SEQ ID NO:36) and RMCA12 (5'-CCGGGCAGAAAG-3') (SEQ ID NO:37) at 657C for two min. followed by cooling to room temperature. 0.5 μg of DNA was ligated to 0.5 nmol of RXMA or RMCA adaptor using T4 DNA ligase (NEB). PCR was performed using 3 μl of each ofthe ligation mix as a template in a 100 μl volume containing 100 pmol of RXA24 or RMC24 primer, 5 units of Taq DNA polymerase, (GIBCO-BRL.), 4 mM MgC12, 16 mM of NH4 (SO4)2, lOmg/ml of BSA, and 5% v/v DMSO. The reaction mixture was incubated at 72 C at 5 min and at 95 C for 3 min. Samples were then subjected to 25 cycles of amplification consisting of 1 min at 95°/C, and 3 min either at 72 C or 777C in a thermal cycler (Hybaid, Inc.). The final extension time was 10 min.
Detection of Aberrant Methylation Using MCA. MCA products from normal colon mucosa and corresponding cancer tissues were prepared as described above. One μg of MCA products was resuspended in 4 μl of TE (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0), mixed with 2 μl of 20 X SSC, and 1 μl aliquot of this mix was blotted onto nylon membranes (Nunc) using a 96 well replication system (Nunc). The membranes were baked at 807C, UN crosslinked for 2 min. and hybridized using 32P labeled probes. Each sample was blotted in duplicate. Each filter included mixtures of a positive control (Caco2) and a negative control (normal colon mucosa from an 18 year old individual). The filters were exposed to a phosphor screen for 24 to 72 hours and developed using a phosphorimager (Molecular Dynamics). The intensity of each signal was calculated using the Image Quant software, and methylation levels were determined relative to the control samples.
RDA.
RDA was performed essentially as previously reported (Lisitsyn et al, 1993) with the following modifications. For the first and second rounds of competitive hybridization, 500 ng and 100 ng of ligation mix was used, respectively. To eliminate the digested adaptor, a cDΝA spun column (Amersham) was used instead of excising from the agarose gel. Primers used for the first and second rounds of RDA are as follows :
Figure imgf000069_0001
After the second round of competitive hybridization, PCR products were digested with Xmal. The J adaptor was eliminated by column filtration. The PCR products were then subcloned into Bluescript SK(-) (Stratagene). To screen for inserts, a total of 396 clones were cultured overnight in LB medium with ampicillin and 3 μl ofthe culture was directly used as template for a PCR reaction. Each clone was amplified with
T3 (5'-AATTAACCCTCACTAAAGGG-3') (SEQ ID NO:46) and T7 (5'-GTAATACGACTCACTATAGGGC-3 ') (SEQ ID NO:47) primers, blotted onto nylon membranes, and screened for cross hybridization with 32P labeled inserts. The clones differentially hybridizing to tester and driver MCA products were further characterized by Southern blot analysis and DNA sequencing.
Southern blot analysis.
Five μg of DNA was digested with 20-100 units of restriction enzymes as specified by the manufacturer (NEB). DNA fragments were separated by agarose gel electrophoresis and transfeπed to a nylon membrane (Zeta-probe, Bio-Rad). Filters were hybridized with 32P-labeled probes and washed at 657C with 2X SSC, 0.1 % SDS for 10 min. twice, and 0.1X SSC, 0.1 % SDS for 20 min. Filters were then exposed to a phosphor screen for 24-72 hours and analyzed by using a phosphorimager (Molecular Dynamics).
DNA sequencing and analysis.
Plasmid DNA was prepared using the Wizard Plus Minipreps (Promega) according to the suppliers recommendation. Sequence analysis was carried out at the Johns Hopkins Core Sequencing Facility using automated DNA sequencers (Applied Biosystems). Sequence homologies were identified using the BLAST program ofthe National Center for Biotechnology Information (NCBI) available at http://www.ncbi.nlm.nih.gov/BLAST using the default parmaters ofthe web site. Putative promoter sequences were predicted using the computer programs NNPP and TSSG available through the Baylor college of Medicine launcher at http://dot.imgen.bcm.tmc.edu:9331.
Bisulfite-restriction methylation analysis.
DNA from colon tumors, cell lines and normal colon mucosa was treated with bisulfite as reported previously (Herman et al, 1996). Primers used for PCR were as follows: hMLHI , 5 '-TAGTAGTYGTTTTAGGGAGGGA-3 ' (SEQ ID
NO:44),
5'-TCTAAATACTCAACRAAAATACCTT-3' (SEQ ID NO:45);
MINTl, 5'-GGGTTGGAGAGTAGGGGAGTT-3' (SEQ ID
NO:46),
5'-CCATCTAAAATTACCTCRATAACTTA-3' (SEQ ID NO:47); MINT2, 5'-YGTTATGATTTTGTTTAGTTAAT-3' (SEQ ID
NO:48), 5 ' -TACACCAACTACCCAACTACCTC-3 ' (SEQ ID NO:409); Versican, 5'-TTATTAYGTTTTTTATGTGATT-3' (VI) (SEQ ID NO:50), 5'-ACCTTCTACCAATTACTTCTTT-3' (V2) (SEQ ID NO:51).
Ten to 20 μl ofthe amplified products were digested with restriction enzymes which distinguish methylated from unmethylated sequences as reported previously (Sadri et al, 1996; Xiong et al, 1997), electrophoresed on 3 % agarose or 5% acrylamide gels, and visualized by ethidium bromide staining.
RT-PCR
Total RNA was prepared from normal colon epithelium and tumor cell lines using TRIZOL (GIBCO-BRL). To study gene expression following demethylation, cell lines were treated with 1 M of 5-aza-2y-deoxycytidine for 2-5 days. cDNA was prepared using random hexamers and reverse transcriptase as specified by the manufacturer (Boehringer). The expression of versican was determined by RT-PCR using the primers
VF 5'-GCTGCCTATGAAGATGGATTTGAGC-3' (SEQ ID
NO:52) and VR 5'-GGAGTTCCCCCACTGT-TGCCA-3' (SEQ ID NO:53).
The PCR products were visualized by ethidium bromide staining. The cDNA samples were also amplified using GAPDH gene, primers
GAPF 5'-CGGAGTCAACGGATTGGTCGTAT-3' (SEQ ID NO:54) and
GAPR 5'-AGCCTTCTCCATGGTGGTGAAGAC-3' (SEQ ID
NO:55) as a control for RNA integrity. All reactions were performed using RT (-) controls where the reverse transcriptase enzyme was omitted.
Chromosomal mapping.
The chromosomal location of clones that did not coπespond to known genes was determined using a human-rodent somatic cell hybrid panel and a radiation hybrid panel (Research Genetics). PCR reactions were performed using 30 ng of each ofthe hybrid panel DNA as a template in a 40 μl volume containing 15 pmol of each primer, 0.5 units of Taq DNA polymerase, (GIBCO BRL), 2mM MgC12, BSA and 5% DMSO. First denaturation was carried out at 957C for 3 min. Samples were then subjected to 35 cycles of amplification consisting of 25 sec. at 947C, 1 min at 60 to 687C and 1.5 min. at 727C in a thermal cycler (Hybaid). The final extension time was 10 min. Ten μl ofthe PCR product were electrophoresed in a 2 % agarose and the genotype of each panel was determined. Linkage analysis was performed using the RH server of Stanford University as described (Stewart et al. , 1997).
Although the invention has been described with reference to the presently prefeπed embodiment, it should be understood that various modifications can be made without departing from the spirit ofthe invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:
A method for identifying a methylated CpG-containing nucleic acid, comprising a) contacting a nucleic acid sample suspected of containing a CpG-containing nucleic acid with a methylation sensitive restriction endonuclease, under conditions and for a time to allow cleavage ofthe nucleic acid; b) contacting the sample with an isoschizomer of said methylation sensitive restriction endonuclease, wherein said isoschizomer of said methylation sensitive restriction endonuclease cleaves both methylated and unmethylated CpG sites. c) adding oligonucleotides to the nucleic acid sample under conditions and for a time to allow ligation ofthe oligonucleotides to the nucleic acid cleaved by said restriction endonuclease; and d) amplifying said cleaved nucleic acid.
2. The method of claim 1, wherein said methylation sensitive restriction endonuclease is Smal.
3. The method of claim 1 , wherein said amplifying is by polymerase chain reaction amplification.
4. The method of claim 3, wherein said amplifying by polymerase chain reaction amplification comprises annealing primers complementary to said oligonucleotide.
5. The method of claim 1, wherein said oligonucleotide comprises a sequence as set forth in a member ofthe group selected from SEQ ID NO:34 (RXMA24) and SEQ ID NO:35 (RXMA12).
6. The method of claim 1, wherein said oligonucleotide comprises a sequence as set forth in a member ofthe group selected from SEQ ID NO:36 (RMCA24) and SEQ ID NO:37 (RMCA12).
7. The method of claim 1 , further comprising adhering the amplified nucleic acid to a membrane.
8. The method of claim 7, further comprising hybridizing the membrane with a probe of interest.
9. The method of claim 1 , wherein the CpG containing nucleic acid comprises a methylated CpG island.
10. The method of claim 9, wherein the CpG island comprises a CpG island located in a gene selected from the group consisting of a pi 6, a Rb, a VHL, a hMLHI, and a BRCAl gene.
11. The method of claim 1 , wherein said sample is selected form the group consisting of a brain cell, a colon cell, a urogenital cell, a lung cell, a renal cell, a hematopoietic cell, a breast cell, a thymus cell, a testis cell, an ovarian cell, a uterine cell, an intestinal cell, serum, urine, saliva, cerebrospinal fluid, pleural fluid, ascites fluid, sputum, and stool.
12. The method of claim 1 , wherein the presence of methylated CpG - containing nucleic acid in the sample is indicative of a cell proliferative disorder.
13. The method of claim 12, wherein the cell proliferative disorder is selected from the group consisting of colon cancer, lung cancer, renal cancer, leukemia, breast cancer, prostate cancer, uterine cancer, astrocytoma, glioblastoma, and neuroblastoma.
14. The method of claim 1 , further comprising performing representation difference analysis, wherein said representation difference analysis comprises hybridizing a driving nucleic acid as a driver.
15. The method of claim 14, wherein said representation difference analysis uses nucleic acid isolated from a member ofthe group consisting of normal colon, normal lung, normal kidney, normal blood cells, normal breast, normal prostate, normal uterus, normal astrocytes, normal glial and normal neurons.
16. A nucleic acid identified by the method of claim 1.
17. A vector comprising the nucleic acid of claim 16.
18. A method for detecting an age-associated disorder, associated with methylation of CpG islands, in a nucleic acid sequence of interest in a subject having or at risk of having said disorder, comprising: a) contacting a nucleic acid sample suspected of comprising a CpG- containing nucleic acid with a methylation sensitive restriction endonuclease, under conditions and for a time to allow cleavage ofthe nucleic acid; b) contacting the sample with an isoschizomer of said methylation sensitive restriction endonuclease, wherein said isoschizomer of said methylation sensitive restriction endonuclease cleaves both methylated and unmethylated CpG-sites, under conditions and for a time to allow cleavage of methylated nucleic acid; c) adding oligonucleotides to the nucleic acid sample under conditions and for a time to allow ligation ofthe oligonucleotides to nucleic acid cleaved by said restriction endonuclease; d) amplifying said cleaved nucleic acid; e) adhering the amplified digested nucleic acid to a membrane; and f) hybridizing the membrane with a probe of interest.
19. The method of claim 18, wherein the sample is selected form the group consisting of brain cells, colon cells, urogenital cell, lung cells, renal cells, hematopoietic cells, breast cell, thymus cells, testis cells, ovarian cells, uterine cells, serum, urine, saliva, cerebrospinal fluid, pleural fluid, ascites fluid, sputum, and stool.
20. The method of claim 18, wherein the probe of interest is a nucleic acid sequence.
21. The method of claim 18, wherein the nucleic acid sequence is selected from the group consisting of a pi 6, a Rb, a VHL, a hMLHI, and a BRCAl nucleic acid.
22. The method of claim 21 , wherein said nucleic acid sequence is a p 16 nucleic acid sequence.
23. The method of claim 18, wherein the sample is a tissue sample or a biological fluid sample.
24. The method of claim 18, wherein the probe is detectably labeled.
25. The method of claim 24, wherein the label is selected from the group consisting of a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, and an enzyme.
26. The method of claim 18, wherein said age-associated disorder is selected from the group consisting of atherosclerosis, diabetes melitis, and dementia.
27. The method of claim 18, wherein said age-associated disorder is a cell proliferative disorder.
28. The method of claim 18, wherein the nucleic acid of interest is a member ofthe group consisting of SEQ ID NOs:l-33 (MINT 1-33).
29. The method of claim 27, wherein said cell proliferative disorder is selected from the group consisting of colon cancer, lung cancer, renal cancer, leukemia, breast cancer, prostate cancer, uterine cancer, astrocytoma, glioblastoma, and neuroblastoma.
30. The method of claim 18, further comprising performing representation difference analysis, wherein said representation difference analysis comprises hybridizing a driving nucleic acid as a driver.
31. The method of claim 30, wherein said representation difference analysis uses nucleic acid isolated from a member ofthe group consisting of normal colon, normal lung, normal kidney, normal blood cells, normal breast, normal prostate, normal uterus, normal astrocytes, normal glial and normal neurons.
32. A method for determining the response of a cell to an agent, comprising: a) contacting a nucleic acid sample suspected of comprising a CpG-containing nucleic acid from said cell with a methylation sensitive restriction endonuclease, under conditions and for a time to allow cleavage of unmethylated nucleic acid; b) contacting the sample with an isoschizomer of said methylation sensitive restriction endonuclease, wherein said isoschizomer of said methylation sensitive restriction endonuclease cleaves methylated and unmethylated CpG-sites, under conditions and for a time to allow cleavage of methylated nucleic acid; c) adding an oligonucleotide to the nucleic acid sample under conditions and for a time to allow ligation ofthe oligonucleotide to nucleic acid cleaved by said restriction endonuclease; d) amplifying said cleaved nucleic acid; e) adhering the amplified cleaved nucleic acid to a membrane; and f) hybridizing the membrane with a probe of interest.
33. The method of claim 32, further comprising performing representation difference analysis, wherein said representation difference analysis comprises hybridizing a nucleic acid as a driver.
34. The method of claim 32, wherein the agent is selected from the group consisting of peptide, peptidomimetic, chemical compound, and a pharmaceutical compound.
35. The method of claim 32, wherein said agent is a chemotherapeutic agent.
36. The method of claim 32, wherein said methylation sensitive restriction endonuclease is Smal.
37. The method of claim 32, wherein said amplifying is by polymerase chain reaction amplification.
38. The method of claim 37, wherein said amplifying by polymerase chain reaction amplification comprises annealing primers complementary to said oligonucleotide.
39. The method of claim 32, wherein said oligonucleotide comprises a sequence as set forth in a member ofthe group selected from SEQ ID NO:34 (RXMA24) and SEQ ID NO:35 (RXMA12).
40. The method of claim 32, wherein said oligonucleotide comprises a sequence as set forth in a member ofthe group selected from SEQ ID NO:36 (RMCA24) and SEQ ID NO:37 (RMCA12).
41. The method of claim 32, wherein said cell is selected form the group consisting of a brain cell, a colon cell, an intestinal cell, a urogenital cell, a lung cell, a renal cell, a hematopoietic cell, a breast cell, a thymus cell, a testis cell, an ovarian cell, a uterine cell, an exocrine cell, and an endocrine cell.
42. A kit useful for the detection of a methylated CpG-containing nucleic acid comprising carrier means containing one or more containers comprising a container containing oligonucleotides for ligation to nucleic acid, a second container containing a methylation sensitive restriction endonuclease and a third container containing an isoschizomer ofthe methylation sensitive restriction endonuclease.
43. The kit of claim 42, wherein said oligonucleotides comprises a sequence as set forth in a member ofthe group selected from SEQ ID NO:34 (RXMA24) and SEQ ID NO:35 (RXMA12).
44. The kit of claim 42, wherein said oligonucleotide comprises a sequence as set forth in a member ofthe group selected from SEQ ID NO:36 (RMCA24) and SEQ ID NO:37 (RMCA12).
45. The kit of claim 42, further comprising one or more containers comprising a primer complementary to said oligonucleotide.
46. A kit useful for the detection of a methylated CpG-containing nucleic acid comprising a carrier means containing one or more containers comprising a membrane, wherein said membrane has a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 (MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32, and MINT33 immobilized on said membrane.
47. An isolated nucleic acid comprising a member selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 (MINTl, MINT2, MINT4, MINT6, MINT8, MINT 9, MINTIO, MINT14, MINTl 5, MINTl 7, MINTl 9, MINT20, MINT22, MINT23, MINT24, MINT27, MINT30, MINT31, MINT32, and MINT33), and degenerate variants thereof.
48. The nucleic acid of claim 47, wherein said nucleic acid is methylated.
49. The nucleic acid of claim 48, wherein said nucleic acid is unmethylated.
50. An substantially purified polypeptide encoded by the nucleic acid of claim 47.
51. The nucleic acid of claim 47, wherein said nucleic acid is operatively linked to an expression control sequence.
52. The nucleic acid of claim 51 , wherein the expression control sequence is a promoter.
53. The nucleic acid of claim 52, wherein the promoter is tissue specific.
54. An expression vector containing the nucleic acid of claim 47.
55. The vector of claim 54, wherein the vector is a plasmid.
56. The vector of claim 54, wherein the vector is a viral vector.
57. The vector of claim 56, wherein the viral vector is a retroviral vector.
58. A host cell containing the vector of claim 54.
59. The host cell of claim 58, wherein the cell is a eukaryotic cell.
60. The host cell of claim 58, wherein the cell is a prokaryotic cell.
61. An isolated nucleic acid sequence comprising a methylated nucleic acid having a sequence as set forth in a member ofthe group consisting of SEQ ID NOs:l-33.
62. A method of identifying a compound that affects methylation of a nucleic acid, comprising: a) incubating components comprising the compound and a sample comprising a nucleic acid sequence identified by the method of claim 1 under conditions sufficient to allow the components to interact; and b) determining the effect ofthe compound on expression ofthe nucleic acid sequence.
63. The method of claim 62, wherein said sample is a cell.
64. The method of claim 62, wherein said sample is a substantially purified nucleic acid.
65. The method of claim 62, wherein the compound is selected from the group consisting of a peptide, a peptidomimetic, a chemical compound, and a pharmaceutical compound.
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EP1127157A1 (en) 2001-08-29
US7700324B1 (en) 2010-04-20
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